CN115066611A

CN115066611A - Multiplex immunofluorescence detection of target antigens

Info

Publication number: CN115066611A
Application number: CN202180013766.1A
Authority: CN
Inventors: 彼得·罗德里克·邓巴; 萨默尔·帕克
Original assignee: Auckland Uniservices Ltd
Current assignee: Auckland Uniservices Ltd
Priority date: 2020-02-28
Filing date: 2021-02-25
Publication date: 2022-09-16
Also published as: AU2021226178A1; EP4111195A4; CA3170429A1; EP4111195A1; WO2021171220A1; US20230095395A1

Abstract

A method of multispectral immunofluorescence imaging of a biological sample is described. The method allows the simultaneous direct detection of seven or more target antigens using directly labeled antibody fluorophore conjugates. The methods enable the simultaneous multiplexed detection and analysis of multiple biomarkers across a planar biological sample, providing unique spatiotemporal insights in immunotherapy and immunodiagnosis.

Description

Multiplex immunofluorescence detection of target antigens

Technical Field

The present invention relates generally to the simultaneous visualization of multiple target antigens in a biological sample, including methods and reagents related thereto.

Background

Multispectral/high-dimensional imaging is of increasing importance to practitioners of biomedical research and clinical medicine/pathology. The ability to visualize multiple specific molecules in a tissue sample provides a powerful tool for research and clinical medical applications. For example, this ability allows the spatial arrangement of different cell types to be determined, with applications for health and disease management and treatment.

In medicine, there is a need to detect target molecules, particularly proteins, in tissue samples for use as biomarkers or "biomarkers" to help identify the type of therapy to which a patient may respond. In one example, patients with cancer in a particular tissue may be grouped for different therapies based on the biomarkers detected in that tissue. In pathology, it may also be desirable to quantify the number of cells expressing a particular target protein before recommending a particular therapy.

Currently used immunohistochemical techniques will typically detect a single target protein in any one tissue section of a tissue sample. For many diseases (e.g. breast cancer), this means that several tissue sections must be labeled with different antibodies before the optimal therapy that can be performed is recommended.

The immunofluorescence microscopy (IFM) technique currently employed allows for the simultaneous detection of more than one molecule in a single tissue section of a sample. However, current technology typically uses indirect labeling with unconjugated primary antibody, followed by labeling of the primary antibody with different secondary antibodies conjugated to different Fluorophores (FP), each having a different emission spectrum. This secondary labeling process allows for the separate detection and localization of target proteins labeled by various antibodies in the same tissue section.

Unfortunately, while IFMs have been used in biomedical research and in some clinical pathology settings, IFMs are generally limited to detecting only two to three, up to four different colors; that is, two to three different antibody tags and nuclear stain are detected. This limitation is due to the ability of conventional fluorescence microscopes to separate only certain emission spectra of different fluorophores.

Currently, the use of multispectral imaging has increased the number of fluorophores that can be distinguished in a single tissue section. For example, the Opal staining platform can label up to 9 different target antigens in a single section. However, there are significant drawbacks associated with the use of current IFM labeling schemes in multispectral, multiplexed imaging of tissue sections, including the labor and time required to complete the scheme.

Current multiple labeling techniques are labor intensive. For example, Opal staining techniques typically require 3-5 days to complete multicolor staining. These platforms are also difficult to iterate, which means that developing new multi-color staining protocols will often take months. With respect to Opal, developing new and/or improved panels (panel) requires considerable knowledge of the IFM and the specific markers of interest.

Thus, there is a need in both research and clinical medicine for new and improved methods for multiplex immunofluorescence detection of multiple target molecules from a single tissue sample.

It is an object of the present invention to at least address some of the above-mentioned deficiencies of the prior art, and/or to at least provide the public with a useful choice, by providing a method for multiplex immunofluorescence detection of multiple target molecules from a single tissue sample, including reagents used therein.

In this specification, reference has been made to patent specifications, other external documents, or other sources of information, which are generally intended to provide a context for discussing the features of the invention. Unless otherwise expressly stated, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

Disclosure of Invention

In one aspect, the invention relates to a composition comprising at least three, four, five, six, or at least seven antibody-fluorophore conjugates (Ab-FP), wherein each FP has a different maximum fluorescence excitation and emission wavelength (Ex).

In another aspect, the invention relates to a method for direct immunofluorescence analysis of a biological sample comprising

a) Labeling at least one target antigen in a planar sample of a biological sample with at least one unique Ab-FP conjugate, and

b) generating a multispectral fluorescence image of the labeled planar sample using a multispectral scanner, wherein the image comprises at least two colors, wherein at least one color is associated with specific binding of at least one unique Ab-FP conjugate and at least one target antigen, and

c) determining from the image the presence or absence of a biomarker comprising at least one target antigen.

In another aspect, the invention relates to a multispectral immunofluorescence image of a planar biological sample, the image comprising at least three, preferably four, five, six, seven, preferably at least eight colors, wherein the at least three, preferably four, five, six, preferably seven colors are associated with specific binding of the at least three, preferably four, five, six, preferably seven Ab-FP and a target antigen comprised in the planar sample.

In another aspect, the invention relates to a method of detecting multiple target antigens in a biological sample comprising

a) Simultaneously labeling at least two target antigens in a planar sample of a biological sample with at least two unique Ab-FP conjugates, wherein the at least two target antigens are present on or in cells in the sample, an

b) Generating a multispectral fluorescence image of the labeled planar sample using a multispectral scanner, wherein the image comprises at least three colors, wherein at least two colors are associated with specific binding of each unique Ab-FP conjugate to the target antigen, and

c) the presence or absence of at least two target antigens, each labeled with a different unique Ab-FP, is determined from the image.

In another aspect, the invention relates to a method of detecting multiple biomarkers in a biological sample comprising

a) Simultaneously labeling at least two target antigens in a planar sample of a biological sample with at least two unique Ab-FP conjugates, wherein a biomarker in the sample comprises each target antigen, and

b) generating a multispectral fluorescence image of the labeled planar sample using a multispectral scanner, wherein the image comprises at least three colors, wherein at least two colors are associated with the specific binding of each unique Ab-FP conjugate and the target antigen, and

c) the presence or absence of a plurality of biomarkers is determined from the image, each biomarker comprising a target antigen labeled with a different unique Ab-FP.

In another aspect, the invention relates to a method of detecting a plurality of different cell types in a biological sample comprising

a) Simultaneously labeling at least two target antigens in a planar sample with at least two unique Ab-FP conjugates, wherein the target antigens are present on or in cells in the sample, and

b) generating a multispectral fluorescence image of the labeled planar sample using a multispectral scanner, wherein the image comprises at least three colors, wherein at least two colors are associated with specific binding of each unique Ab-FP conjugate to a target antigen on different cell types, and

c) the presence or absence of at least two cell types, each labeled with a different unique Ab-FP, is determined from the image.

In another aspect, the invention relates to a method of identifying the abundance of a plurality of cell types in a biological sample, comprising:

a) simultaneously labeling a planar biological sample with at least two, preferably three, four, five, six, preferably at least seven unique antibody-fluorophore conjugates (Ab-FPs), wherein each Ab-FP specifically binds to a target antigen on or in a different cell, and

b) generating a multispectral image of the labeled planar sample by simultaneously detecting the fluorescence emission spectra of each FP from each Ab-FP, respectively, and

c) determining the abundance of the plurality of different cell types in the planar sample based on the detected fluorescence emission spectra, optionally according to a suitable reference control.

In another aspect, the invention relates to a method of determining the spatial distribution of a plurality of cell types in a biological sample, comprising

a) Simultaneously labeling at least two target antigens in a planar sample of a biological sample with at least two unique Ab-FP conjugates, wherein each of the at least two target antigens is present on or in a different cell type in the sample,

b) generating a multispectral fluorescence image of the labeled planar sample using a multispectral scanner, wherein the image comprises at least three colors, wherein at least two colors are associated with the specific binding of each unique Ab-FP conjugate to the target antigen,

c) identifying at least two different cell types from the image based on the binding of each Ab-FP and at least two target antigens, an

d) The spatial distribution of a plurality of cell types in a biological sample is determined from the image.

In another aspect, the invention relates to a method of identifying a subset of patients from a group of patients, comprising:

a) simultaneously labeling at least three, preferably four, five, six, preferably seven different biomarkers in a planar biological sample from a patient with at least two, preferably three, four, five, six, preferably seven unique antibody-fluorophore conjugates (Ab-FP), wherein each Ab-FP specifically binds to a target antigen on a biomarker,

b) generating a multispectral image of the labeled planar sample portion by simultaneously detecting the fluorescence emission spectra of each FP in each Ab-FP,

c) detecting the presence or abundance of each biomarker in the image generated in b), wherein each biomarker is identified in the image as a different color associated with specific binding of a unique Ab-FP, and

d) the patients are determined from the images to be in a subgroup based on the presence or abundance of each Ab-FP that specifically binds to each biomarker.

In another aspect, the invention relates to a method of making a diagnostic panel of antibody-fluorophore conjugates (Ab-FP), comprising:

a) identifying at least three, preferably four, five, six, preferably seven biomarkers of a predetermined disease or condition,

b) obtaining unique Ab-FPs for each identified biomarker, each Ab-FP including an antibody that specifically binds to a target antigen on one of the biomarkers identified in a), preferably each biomarker identified in a), each Ab-FP having a Fluorophore (FP) with a wavelength of maximum fluorescence emission from about 420nm to about 850nm,

c) simultaneously labeling the planar biological sample with the Ab-FP of b), wherein labeling comprises specifically binding each Ab-FP to a biomarker, preferably wherein each Ab-FP binds a different biomarker separately,

d) a multispectral image of the fluorescence emission spectrum of each FP was obtained,

e) identifying the presence or abundance of each biomarker in the multispectral image, wherein each biomarker is identified in the image as a different color associated with specific binding of a different Ab-FP and the target antigen on the biomarker, and

f) selecting a unique Ab-FP conjugate that can be identified in the image in e) as a set of Ab-FPs for diagnosis of the disease or condition predetermined in a).

In another aspect, the invention relates to a method of identifying an alternative antibody-fluorophore (Ab-FP) conjugate for direct immunofluorescence analysis of a biological sample, comprising:

a) generating a first multispectral immunofluorescence image from a single planar biological sample using a first set of at least two to at least seven unique Ab-FP conjugates, the first multispectral immunofluorescence image comprising up to eight different colors, wherein up to seven colors are each associated with a unique Ab-FP conjugate according to the methods described herein,

b) selecting at least one of the Ab-FP conjugates from the first set for replacement with a replacement Ab-FP,

c) identification of suitable antibodies for replacement of Ab-FP

d) Identification of suitable fluorophores for replacement of Ab-FP

e) The replacement Ab-FP was obtained,

f) replacing the selected Ab-FP in b) with a replacement Ab-FP to produce a second set of at least two to at least seven unique Ab-FP conjugates,

g) generating a second immunofluorescence image using a second set of Ab-FP conjugates according to the methods described herein, an

h) Comparing the first multispectral immunofluorescence image and the second multispectral immunofluorescence image,

wherein the ability to distinguish between each different color in the multispectral image is not different when the first image and the second image are compared in h), confirming the identification of the replacement Ab-FP conjugate.

In another aspect, the invention relates to a method for determining whether at least one cell type is responsive to a drug candidate; the method comprises the following steps:

a) determining the abundance or spatial distribution of at least one cell type in a planar biological sample comprising the at least one cell type using the multispectral immunofluorescence detection method described herein, and

b) determining whether the at least one cell type is responsive to the drug candidate based on the abundance or spatial distribution of the at least one cell type in the sample, optionally compared to a suitable control.

In one embodiment, the planar biological sample comprises cells cultured in vitro. In one embodiment, the planar biological sample comprises a biological tissue or a portion thereof.

In another aspect, the invention relates to a method for predicting a patient's response to treatment with a proposed treatment for a predetermined disease or condition, the method comprising:

a) determining the abundance or spatial distribution of at least one cell type in a planar biological sample from a patient using the multispectral immunofluorescence detection method described herein, and

b) determining that the patient will or will not respond to the proposed treatment based on the abundance or spatial distribution of the at least one cell type in the sample, optionally compared to a suitable control.

In another aspect, the invention relates to a method for identifying a cellular response to a drug candidate comprising

a) Contacting a planar biological sample containing a plurality of cells with a drug candidate,

b) determining the abundance or spatial distribution of at least one cell type in a sample using the multispectral immunofluorescence detection methods described herein, and

c) determining the presence of a cellular response to the candidate drug from the abundance or spatial distribution of the at least one cell type in the sample, optionally compared to a suitable control.

In another aspect, the invention relates to a method of identifying a patient who would benefit from a candidate therapy comprising:

a) labeling a planar biological sample obtained from a subject with at least one unique Ab-FP conjugate, preferably with from 1 to 12 unique Ab-FP conjugates,

b) obtaining at least one digital fluorescence image of the labeled sample using a multispectral scanner;

c) extracting data associated with at least one emission spectrum associated with the Ab-FP conjugate,

d) calculating a distribution function that captures a data distribution of at least one emission spectrum;

e) deriving an overall score for the patient from the distribution function;

f) evaluating the total score against at least one reference value; selecting the subject as a candidate for the indicated therapy based on the total score, an

g) The subject is optionally treated with a specified therapy.

a) Simultaneously labeling at least seven target antigens in a planar sample of a biological sample with at least two unique Ab-FP conjugates, wherein a biomarker in the sample comprises each target antigen, and

b) generating a multispectral fluorescence image of the labeled planar sample using a multispectral scanner, wherein the image comprises at least eight colors, wherein at least seven colors are associated with the specific binding of each unique Ab-FP conjugate to the target antigen, and

c) the presence or absence of multiple biomarkers is determined from the image, each biomarker comprising a target antigen labeled with a different unique Ab-FP.

The various embodiments of the different aspects of the invention discussed above are also set forth in the detailed description of the invention below, but the invention is not limited thereto.

Other aspects of the invention will become apparent from the following description which is given by way of example only and with reference to the accompanying drawings.

Drawings

The invention will now be described with reference to the figures in the accompanying drawings.

FIG. 1-demixed image (grayscale) showing CD 3-expressed Ab-FP + DAPI-labeled melanoma infiltrating lymph node tissue

A de-blended image of one of the seven colors (6 Ab-FP + DAPI) detected simultaneously from Ab-FP labeled melanoma infiltrating lymph node tissue sections, showing only the distribution of CD3+ T cells. CD3+ T cells labeled with anti-CD 3-AF532 antibody conjugate appear bright and/or gray.

FIG. 2-demixed image (grayscale) showing Ab-FP + DAPI-labeled melanoma infiltrating lymph node tissue expressed by CD21

A de-blended image of one of the seven colors (6 Ab-FP + DAPI) detected simultaneously from Ab-FP labeled melanoma infiltrating lymph node tissue sections, showing only the distribution of CD21+ B cells and follicular dendritic cells. CD21+ B cells and follicular dendritic cells labeled with anti-CD 21-BB700 antibody conjugate appear bright and/or gray.

FIG. 3-demixed image (grayscale) showing Ab-FP + DAPI-labeled melanoma infiltrating lymph node tissue expressed by CD31

A de-blended image of one of the seven colors (6 Ab-FP + DAPI) detected simultaneously from Ab-FP labeled melanoma infiltrating lymph node tissue showed only CD31+ blood and lymphatic endothelial cell distribution. CD31+ blood and lymphatic endothelial cells labeled with anti-CD 31-BV480 antibody conjugate appear bright and/or gray.

FIG. 4-demixing image (grayscale) showing Ab-FP + DAPI-labeled CD 34-expressing melanoma infiltrating lymph node tissue

A de-blended image of one of the seven colors (6 Ab-FP + DAPI) detected simultaneously from Ab-FP labeled melanoma infiltrating lymph node tissue sections, showing only the distribution of CD34+ blood and lymphatic endothelial cells. CD34+ blood and lymphatic endothelial cells labeled with anti-CD 34-PE-CF594 antibody conjugate appear bright and/or gray.

FIG. 5-demixing image (grayscale) showing CD 141-expressed Ab-FP + DAPI-labeled melanoma-infiltrating lymph node tissue

A de-blended image of one of the seven colors (6 Ab-FP + DAPI) detected simultaneously from Ab-FP labeled melanoma infiltrating lymph node tissue sections, showing only the distribution of CD141+ limbic reticulocytes and dendritic cells. CD141+ limbic reticulocytes and dendritic cells labeled with anti-CD 141-BB515 antibody conjugate appear bright and/or gray.

FIG. 6-demixed image (grayscale) showing Ab-FP + DAPI-labeled, Ki 67-expressed melanoma infiltrating lymph node tissue

A de-blended image of one of the seven colors (6 Ab-FP + DAPI) detected simultaneously from Ab-FP labeled melanoma infiltrating lymph node tissue sections showed only a distribution of Ki67+ cells. Ki67+ cells labeled with anti-Ki 67-AF647 antibody conjugate appear bright and/or gray.

FIG. 7-demixing image (grayscale) of Ab-FP + DAPI-labeled melanoma infiltrating lymph node tissue showing only the nuclear stain DAPI

A de-blended image of one of seven colors (6 Ab-FP + DAPI) detected simultaneously from Ab-FP labeled melanoma infiltrated lymph node tissue sections, showing only the nuclear stain DAPI.

FIG. 8-Ab-FP + DAPI-labeled Combined image (grayscale) of melanoma-infiltrating lymph node tissue.

A combination of unmixed images in fig. 1 to 7, showing all seven colors (6 Ab-FP + DAPI) detected simultaneously from Ab-FP labeled melanoma-infiltrated lymph node tissue sections.

FIG. 9-Combined image (grayscale) of Ab-FP labeled melanoma infiltrating lymph node tissue without DAPI.

Combinations of unmixed images in FIGS. 1 to 6, showing six of seven colors (6 Ab-FP + DAPI) detected simultaneously from Ab-FP labeled melanoma infiltrating lymph node tissue sections. DAPI is not shown.

FIG. 10-Ab-FP + DAPI-labeled Combined image (color) of melanoma-infiltrating lymph node tissue.

A combination of unmixed images in fig. 1 to 7, showing all seven colors (6 Ab-FP + DAPI) detected simultaneously from Ab-FP labeled melanoma-infiltrating lymph node tissue sections.

FIG. 11-Combined image (color) of Ab-FP labeled melanoma infiltrating lymph node tissue without DAPI.

FIG. 12-Ab-FP labeled melanoma infiltrating lymph node tissue + DAPI combined image (grayscale).

Combined unmixed images of seven colors (6 Ab-FP + DAPI) detected simultaneously from Ab-FP labeled melanoma infiltrating lymph node tissue sections. As shown in figures 1 to 6 respectively, the images show the distribution of T cells, B cells, FDCs (follicular dendritic cells), MRCs (marginal reticulocytes), BECs (blood endothelial cells), LECs (lymphatic endothelial cells) and Ki67+ cells. DAPI represents nuclear stain.

Fig. 13-combined image (grayscale) of Ab-FP labeled melanoma-infiltrating lymph node tissue without DAPI.

A combined unmixed image of six of seven colors (6 Ab-FP + DAPI) detected simultaneously from Ab-FP labeled melanoma infiltrating lymph node tissue sections. As shown in fig. 1 to 6, respectively, the images show the distribution of T cells, B cells, FDC (follicular dendritic cells), MRC (marginal reticulocytes), BEC (blood endothelial cells), LEC (lymphatic endothelial cells) and Ki67+ cells. DAPI is not shown.

FIG. 14-demixed image (grayscale) showing only Ab-FP + DAPI-labeled melanoma-infiltrating lymph node tissue of DAPI.

A de-blended image of one of the eight colors (7 Ab-FP + DAPI) detected simultaneously from Ab-FP labeled melanoma infiltrating lymph node tissue sections, showing only the nuclear stain DAPI.

FIG. 15-demixed image (grayscale) showing CD 31-expressed Ab-FP + DAPI-labeled melanoma infiltrating lymph node tissue.

A de-blended image of one of the eight colors (7 Ab-FP + DAPI) detected simultaneously from Ab-FP labeled melanoma infiltrating lymph node tissue sections, showing only the distribution of CD31+ blood and lymphatic endothelial cells. CD31+ blood and lymphatic endothelial cells labeled with anti-CD 31-BV480 antibody conjugate appear bright and/or gray.

FIG. 16-shows a demixed image (grayscale) of CD 141-expressed Ab-FP + DAPI-labeled melanoma-infiltrating lymph node tissue.

A de-blended image of one of the eight colors (7 Ab-FP + DAPI) detected simultaneously from Ab-FP labeled melanoma infiltrating lymph node tissue sections, showing only the distribution of CD141+ limbic reticulocytes and dendritic cells. CD141+ limbic reticulocytes and dendritic cells labeled with anti-CD 141-BB515 antibody conjugate appear bright and/or gray.

FIG. 17-demixed image (grayscale) showing CD 3-expressed Ab-FP + DAPI-labeled melanoma infiltrating lymph node tissue.

A de-blended image of one of the eight colors (7 Ab-FP + DAPI) detected simultaneously from Ab-FP labeled melanoma infiltrating lymph node tissue sections, showing only the distribution of CD3+ T cells. CD3+ T cells labeled with anti-CD 3-AF532 antibody conjugate appear bright and/or gray.

FIG. 18-demixed image (grayscale) showing CD 34-expressed Ab-FP + DAPI-labeled melanoma infiltrating lymph node tissue.

A de-blended image of one of the eight colors (7 Ab-FP + DAPI) detected simultaneously from Ab-FP labeled melanoma infiltrating lymph node tissue sections, showing only the distribution of CD34+ blood and lymphatic endothelial cells. CD34+ blood and lymphatic endothelial cells labeled with anti-CD 34-PE-CF594 antibody conjugate appear bright and/or gray.

Figure 19-shows a demixed image (grayscale) of Ki 67-expressed Ab-FP + DAPI-labeled melanoma-infiltrated lymph node tissue.

A de-blended image of one of the eight colors (7 Ab-FP + DAPI) detected simultaneously from Ab-FP labeled melanoma infiltrating lymph node tissue sections showed only a distribution of Ki67+ cells. Ki67+ cells labeled with anti-Ki 67-AF647 antibody conjugate appear bright and/or gray.

FIG. 20-demixed image (grayscale) showing CD 21-expressed Ab-FP + DAPI-labeled melanoma infiltrating lymph node tissue.

A de-blended image of one of the eight colors (7 Ab-FP + DAPI) detected simultaneously from Ab-FP labeled melanoma infiltrating lymph node tissue sections, showing only the distribution of CD21+ B cells and follicular dendritic cells. CD21+ B cells and follicular dendritic cells labeled with anti-CD 21-BB700 antibody conjugate appear bright and/or gray.

FIG. 21-demixed image (grayscale) showing Ab-FP + DAPI-labeled melanoma infiltrating lymph node tissue expressed by CD11 c.

A de-blended image of one of the eight colors (7 Ab-FP + DAPI) detected simultaneously from Ab-FP labeled melanoma infiltrating lymph node tissue sections, showing only the distribution of CD11c + cells. CD11c + cells labeled with anti-CD 11c-AF700 antibody conjugate appear bright and/or gray.

FIG. 22-Combined image (grayscale) showing Ab-FP + DAPI-labeled melanoma infiltrating lymph node tissue in 8 color combinations.

A combination of unmixed images in fig. 14 to 21, showing all eight colors (7 Ab-FP + DAPI) detected simultaneously from Ab-FP labeled melanoma-infiltrated lymph node tissue sections. The combined demixed images show the distribution of T cells, B cells, FDC (follicular dendritic cells), MRC (marginal reticulocytes), BEC (blood endothelial cells), LEC (lymphatic endothelial cells), CD11c + cells and Ki67+ cells. DAPI represents the nuclear stain.

Fig. 23-combined image (grayscale) showing Ab-FP + DAPI labeled melanoma infiltrating lymph node tissue in 7 color combinations (DAPI not shown).

A combination of unmixed images in fig. 14 to 21, which show seven colors detected simultaneously from Ab-FP labeled melanoma infiltrating lymph node tissue sections (7 Ab-FP + DAPI, where DAPI is not shown). The combined demixed images show the distribution of T cells, B cells, FDC (follicular dendritic cells), MRC (marginal reticulocytes), BEC (blood endothelial cells), LEC (lymphatic endothelial cells), CD11c + cells and Ki67+ cells.

FIG. 24-shows a demixed image of CD 163-expressed melanoma-infiltrated lymph node tissue sections.

Demalending images of melanoma infiltrated lymph node tissue stained with anti-CD 163 APC/Fire 750, which showed CD163+ cell distribution. CD163+ cells labeled with anti-CD 163-APC/Fire 750 antibody conjugate appear light and/or grey.

FIG. 25-shows a demixed image of CD 163-expressed melanoma-infiltrated lymph node tissue sections.

FIG. 26-demixed images (grayscale) showing CD19 expression (with or without DAPI): AF647

Demixed images of 2 color (CD19-AF647+ DAPI) stained FFPE tonsil tissues, which showed distribution of CD19+ B cells (detected by anti-CD 19 AF647 antibody), with (a) or without (B) DAPI.

CD19+ B cells labeled with anti-CD 19-AF647 antibody conjugate appear bright and/or gray.

FIG. 27-demixed images showing CD8 expression (with or without DAPI): AF647

Unmixed images of FFPE tonsil tissue stained with 2 colors (CD8-AF647+ DAPI) showing the distribution of CD8+ T cells (detected by anti-CD 8 AF647 antibody), with (a) or without (B) DAPI.

CD8+ T cells labeled with anti-CD 8 AF647 antibody conjugate appear bright and/or gray.

FIG. 28-demixed images showing CD45RO expression (with or without DAPI): AF488

Demixed images of 2 color (CD45RO-AF488+ DAPI) stained FFPE tonsil tissues, which showed the distribution of CD45RO + activated and memory T cells and some B cells (detected by anti-CD 45RO AF488 antibody), with (a) or without (B) DAPI.

CD45RO + activated T cells, memory T cells, and some B cells labeled with anti-CD 45RO AF488 antibody conjugate appear bright and/or gray.

FIG. 29-demixed images showing CD45RO expression (with or without DAPI): AF594

Unmixed images of 2 color (CD45-AF594+ DAPI) stained FFPE tonsil tissues, showing the distribution of CD45RO + activated and memory T cells and some B cells (detected by anti-CD 45RO F594 antibody), with (a) or without (B) DAPI.

FIG. 30-demixed images showing CD45RO expression (with or without DAPI): AF700

Unmixed images of 2 color (CD45-AF700+ DAPI) stained FFPE tonsil tissues, showing the distribution of CD45RO + activated and memory T cells and some B cells (detected by anti-CD 45RO AF700 antibody), with (a) or without (B) DAPI.

FIG. 31-demixed images showing CD45RO expression (with or without DAPI): BV510

Unmixed images of 2 color (CD45-BV510+ DAPI) stained FFPE tonsil tissues showing the distribution of CD45RO + activated and memory T cells and some B cells (detected by anti-CD 45RO BV510 antibody), with (a) or without (B) DAPI.

FIG. 32-demixed images showing CD45RO expression (with or without DAPI): BV650

Unmixed images of 2 color (CD45-BV650+ DAPI) stained FFPE tonsil tissue showing the distribution of CD45RO + activated and memory T cells and some B cells (detected by anti-CD 45RO BV650 antibody), with (a) or without (B) DAPI.

FIG. 33-demixed images showing CD45RO expression (with or without DAPI): PE/Dazle 594

Unmixed images of 2-color (CD45-PE/Dazzle 594+ DAPI) stained FFPE tonsil tissue showing the distribution of CD45RO + activated and memory T cells and some B cells (detected by anti-CD 45RO PE/Dazzle594 antibody), with (a) or without (B) DAPI.

FIG. 34-demixed images showing CD45RO expression (with or without DAPI): APC/Fire 750

Unmixed images of 2 color (CD45-APC/Fire 750+ DAPI) stained FFPE tonsil tissues showing the distribution of CD45RO + activated T cells and memory T cells and some B cells (detected by anti-CD 45RO APC/Fire 750 antibody), with (A) or without (B) DAPI.

FIG. 35-demixed images showing CD19 and CD45RO expression

A combined unmixed image (a) of 3 colours (2 Ab + DAPI) stained FFPE tonsil tissues, showing the distribution of CD19+ B cells (detected (B) by anti-CD 19-AF647 antibody), and CD45RO + activated T cells and memory T cells and some B cells (detected (C) by anti-CD 45RO AF488 antibody) and DAPI (d).

FIG. 36-demixed images showing CD8 and CD45RO expression

A combined unmixed image (a) of 3 colors (2 Ab + DAPI) stained FFPE tonsil tissue, showing the distribution of CD8+ T cells (detected (B) by anti-CD 8-AF647 antibody), and CD45RO + activated and memory T cells and some B cells (detected (C) by anti-CD 45RO AF488 antibody) and DAPI (d).

All images in fig. 1 to 36 were acquired using Vectra Polaris multi-spectra.

Detailed Description

Definition of

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It is also contemplated that the practice of the present invention may be performed using standard immunological, histological, cell biological, molecular biological, pharmacological and biochemical protocols and procedures known in the art.

The following definitions are provided to better define the invention and to serve as guidance to one of ordinary skill in the art in practicing the invention.

All patents and publications, including all sequences disclosed within such patents and publications, referred to herein are expressly incorporated by reference.

The term "Ab-FP conjugate" (also abbreviated herein as Ab-FP) and grammatical variants thereof, as used herein, refers to an antibody-fluorophore conjugate in which the antibody and fluorophore in the conjugate are directly linked to each other by at least one covalent bond. In some embodiments, the Ab-FP conjugate includes a single Ab moiety having one or more covalently bound FPs. In some embodiments, an Ab-FP includes multiple FPs covalently bound to a single Ab. The antibody in the Ab-FP conjugate is a primary antibody that binds directly to a naturally occurring target antigen on a biomolecule.

The term "unique antibody-fluorophore conjugate" (including grammatical variants and abbreviations thereof) when used with reference to Ab-FP refers to any composition containing a "unique antibody fluorophore conjugate" that includes an antibody and a fluorophore that is different from any other antibody or fluorophore included in the composition or any other Ab-FP that may be used with the composition. A composition comprising at least two distinct Ab-FPs means that the antibody and fluorophore in each of the two Ab-FPs are different from each other. Likewise, a composition comprising at least three unique Ab-FPs means that the antibodies and FPs in each of the three Ab-FPs are different from each other.

The term "maximum fluorescence excitation and emission wavelength (Ex/Em)" and grammatical variations thereof as used herein refers to the maximum excitation (Ex) wavelength (nm) of a fluorophore and the maximum emission (Em) wavelength of the fluorophore.

As used herein, the term "fluorescence excitation and emission spectrum" and grammatical variations thereof refers to a plurality of excitation and emission wavelengths of a given fluorophore that are different from the given fluorophore and are detected using the multispectral scanner described herein.

As used herein, "multispectral scanner" and grammatical variations thereof refer to a device capable of collecting data over a variety of different wavelength ranges, such as described in US 6399299 or US 9006684, which are expressly incorporated herein by reference, including all patents and publications disclosed therein.

The terms "labeled" and "labeling" and grammatical variations thereof as used herein with respect to Ab-FP means that the Ab included in Ab-FP has specifically bound to a target antigen in situ in a planar biological sample under conditions that allow immunofluorescence detection of the FP included in Ab-FP.

The term "antibody" and grammatical variants thereof refer to an immunoglobulin molecule having a specific structure that specifically interacts with (binds to) a molecule that includes its cognate antigen. In some embodiments, the antigen is an antigen used to synthesize an antibody. This antigen is referred to as the target antigen.

The phrase "each Ab is different from each other" means that each Ab specifically binds to a different target antigen.

As used herein, the term "antibody" and grammatical variants thereof broadly includes full-length antibodies, and may also include certain antigen-binding portions and/or fragments thereof. Also included are monoclonal and polyclonal antibodies, multivalent and monovalent antibodies, multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies, human antibodies, humanized antibodies, and affinity matured antibodies and antigen binding portions and/or fragments thereof.

A "target antigen" that is "specifically bound" or "specifically labeled" (including grammatical variants thereof) by an antibody in Ab-FP as described herein is an antigen that preferentially binds to the target antibody, e.g., has less than 25%, or less than 10%, or less than 1%, or less than 0.1% cross-reactivity with non-target antibodies. In some embodiments, the target antigen is a protein antigen.

As used herein, "target antigen" refers to an antigen on a biomolecule in a sample that is directly bound by, and thus specifically labeled by, the antibody portion of Ab-FP described herein. The target antigen used herein is not a primary antibody that can bind to a secondary antibody. According to the present invention and as described herein, such primary antibodies are specifically excluded from the target antigen.

Typically, the binding affinity (dissociation constant (Kd) value) of the target antibody for the antigen or epitope does not exceed 10 ^-6 Or 10 ^-7 M, preferably less than about 10 ^-8 M, more preferably less than about 10 ^-9 M, or 10 ^-10 Or 10 ^-11 Or 10 ^-12 And M. Binding affinity can be assessed using surface plasmon resonance [ see, e.g., US 7531639 or US 6818392, each of which is incorporated herein by reference]。

The term "cell type" and grammatical variations thereof as used herein refers to a group of cells defined by the co-presence of one or more expressed target antigens. As used herein, a "cell type" can be a group of cells of any size that expresses one or more target antigens, such as a population of cells, a sub-population of cells, or a smaller group. As a non-limiting example, the cell type can be a population of immune cells known in the art, such as T cells, B cells, or a sub-population of such cells, such as invariant natural killer T cells (inkts).

The terms "planar sample" and "planar biological sample" and grammatical variations thereof as used herein refer to a two-dimensional sample (also referred to as "cellular material") that is substantially planar, i.e., contains biological material of any combination of cells or biomolecular complexes, organelles, sub-cellular structures, or cellular debris. Planar samples can be obtained by slicing a three-dimensional sample containing cells or cellular material and mounting the slices onto a planar surface. Planar samples may also be obtained by growing or depositing cells or cellular material on a planar surface, or by adsorbing or absorbing cells or cellular material to a planar surface. In a particular embodiment of the invention, the planar biological sample is a tissue section.

The term "color" and grammatical variations thereof that "correlates with specific binding of Ab-FP" is the color that appears in a fluorescence image that directly correlates with the fluorescence emission spectra of the FP in the Ab-FP conjugate when the Ab-FP specifically binds the target antigen in situ in a planar biological sample.

The term "suitable control image" and grammatical variations thereof are well understood by those skilled in the art and refer to an image that has been generated to serve as an acceptable comparison control as will be recognized by those skilled in the art. In one non-limiting example, a suitable control image can be an image of an unlabeled tissue section. In another example, a suitable control image may be an image of a tissue section obtained from an earlier point in time to monitor the progression of a disease or disorder, or from a healthy individual, or from an individual prior to the onset of a disease or after medical treatment, but is not so limited. It is believed that the generation of suitable control images can be performed by a skilled person with reference to the relevant art in conjunction with the methods and reagents provided by the present invention.

The term "biomarker" and grammatical variants thereof are used herein as understood by the skilled artisan and include biomolecules comprising a target antigen, as well as cells or cellular structures or cellular substructures comprising the biomolecule. In some embodiments, the biomolecule is a protein, carbohydrate, lipid, or combination thereof; such as, but not limited to, glycolipids or glycoproteins. In one embodiment, the biomolecule is a protein. In one embodiment, the biomolecule is a protein expressed on or in a cell.

When used descriptively, "biomarker" refers to a biological molecule known to be associated with and/or indicative of a particular biological process, characteristic, object, state, condition, or function. In one non-limiting example, a biomarker is indicative of a cell type, cell function, or cellular process. In some embodiments, the biomarker is a functional marker, e.g., a marker of a cellular process that occurs in a number of different cells. In this case, the relative expression of the biomarkers may allow for differentiation of different cell types, cell structures, and/or cell substructures by sufficient labeling of the biomarkers using Ab-FP described herein to determine the presence and/or abundance of the biomarkers. In one example, a biomarker is associated with a disease state or state of disease progression, but is not so limited.

As used herein, the phrase "known to be associated with … …" with respect to a biomarker and grammatical variants thereof refers to a biomarker that indicates a characteristic, molecule, structure, state, or condition of the organism from which it is measured. In some examples, the presence or absence of a biomarker may indicate any or all of a biological feature, molecule, structure, state, or condition. In other examples, the absolute or relative abundance of a biomarker may be indicative of any or all of a biological feature, molecule, structure, state, or condition.

The term "determining the abundance of a cell type" and grammatical variations thereof refers to determining the absolute number of cells comprising a particular biomarker of interest according to the methods described herein. In some embodiments, determining abundance refers to identifying the number of cells in a multispectral immunofluorescence image of a sample having a fluorescence emission intensity greater than a predetermined background level of autofluorescence of the sample.

The term "determining the relative abundance of a cell type" and grammatical variations thereof refers to determining the percentage of the number of cells comprising a particular biomarker of interest from the total population of cell types present in a planar sample, again according to the methods described herein.

"abundance" refers to the total number of labeled cells. Relative abundance refers to the percentage of labeled cells of a given cell type as a percentage of the total cells of that cell type.

The term "multispectral imaging of an entire tissue section" and grammatical variations thereof refers to the use of a multispectral scanner to scan an entire section of a tissue sample presented on a slide or other carrier supporting the section for labeling and imaging, and the image produced by that scan includes the entirety of the tissue section presented on the slide or carrier.

The term "patient" as used herein is used interchangeably with "subject" and is synonymous. The patient or subject is an animal. Preferably, the animal is a mammal. Preferably, mammals include human and non-human mammals, such as cats, dogs, horses, pigs, cows, sheep, deer, mice, rats, primates (including gorillas, rhesus monkeys, and chimpanzees), possums, and other domestic farm or zoo animals, but are not limited thereto. Preferably, the mammal is a human. When used herein to describe a cell type, disease, or disorder, the term "predetermined" and grammatical variations thereof means that the cell type, disease, or disorder is known and can be selected by a skilled artisan in view of the present disclosure and the art.

The term "clinically relevant level" and grammatical variations thereof as used herein refers to the presence and/or abundance of a biomarker detected according to the methods described herein as deemed clinically feasible by the skilled artisan.

As used herein, the term "high abundance" and grammatical variations thereof for describing a biomarker, cell type, cell structure, or substructure refers to the abundance or relative abundance of the biomarker, cell type, cell structure, or substructure in a sample as deemed clinically feasible by the skilled artisan.

The term "clinically actionable" and grammatical variations thereof as used herein refers to the presence and/or abundance of at least one biomarker in a multi-spectral immunofluorescence image generated according to the methods described herein, wherein the detected presence and/or abundance of the biomarker provides clear and convincing evidence to a clinician that a therapeutic intervention is needed.

The term "unique color" and grammatical variations thereof as used herein refers to a color that specifically corresponds to the spectrum of fluorescent energy emitted from a particular FP among the Ab-FPs described herein.

The term "comprising" as used in the specification and claims means "consisting at least in part of … …"; that is, when interpreting statements in this specification and claims which include "comprising," the features prefaced by this term in each statement all need to be present but other features can also be present. Related terms such as "comprise", "comprises", "comprised" and the like are to be interpreted in a similar manner.

The term "consisting essentially of … …" as used herein refers to the specified materials or steps, as well as those that do not materially affect the basic and novel characteristics of the invention as claimed.

The term "consisting of … …" as used herein refers to the specified material or step of the invention claimed, and does not include any elements, steps or components not specified in the claims.

Reference to a range of values disclosed herein (e.g., 1 to 10) is also intended to include all relevant values within that range (e.g., 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9, and 10) and any rational number range within that range (e.g., 2 to 8, 1.5 to 5.5, and 3.1 to 4.7), and therefore all subranges of all ranges explicitly disclosed herein are explicitly disclosed. These are only examples of what is specifically intended, and all possible combinations of numerical values between the lowest value and the highest value enumerated, should be considered to be expressly stated in this application in a similar manner.

Detailed Description

The inventive technique described herein is based on the determination that the inventors can label multiple target antigens simultaneously in a planar biological sample using multiple Ab-FP conjugates described herein. In one example, the labeled sample is a tissue slice, which can then be imaged using a multispectral scanner and suitable image analysis software to generate a multispectral image, wherein the presence and/or abundance of two to twelve different biomarkers can be determined simultaneously.

The inventors have unexpectedly discovered that specific and simultaneous labeling of multiple biomarkers can be performed using the Ab-FP conjugates described herein. In this way, a multispectral image can be generated that depicts multiple colors, each color associated with the specific binding of Ab-FP, and each color providing specific information related to a particular biomarker.

The ability to simultaneously and rapidly label and directly detect such a large number of biomarkers according to the methods described herein provides clinicians with a powerful research tool with significant, unexpected and unexpected advantages in biomarker detection. In addition, the methods described herein can be used by a skilled artisan for a variety of clinical applications, including disease diagnosis, patient prognosis, and other applications requiring differentiation of multiple cell types in a tissue, e.g., based on the presence and/or abundance of one or more target antigens/biomarkers.

Biomarkers

One skilled in the art recognizes that a biomolecule marker or "biomarker" is a field term that describes a biomolecule (in this case, a protein), the presence of which can be considered to indicate the diagnosis and/or prognosis of a particular cell type and/or a particular biological event or context (e.g., a disease, cell population, or tissue). Detection of one or more "biomarkers" by various means can be used for a variety of research and clinical purposes.

For example, in the present invention, a biomarker can be any target biomolecule, preferably a protein, that is detected and visualized by the specific binding of Ab-FP described herein to a target antigen included on the biomolecule according to the multispectral immunofluorescence detection method described herein. The target antigen is present on a biomolecule, which itself will be present in or on the cell. In this way, biomarkers can be used to detect cells, cell types, cell structures or substructures, but are not limited thereto. In one embodiment, the biomolecule is a protein or an antigen including a portion thereof.

In one exemplary case, the Ab-FP conjugates described herein are used to label a target antigen on CD21 protein, wherein the target antigen is present on the protein, wherein the protein is present on the cell and serves as one of several biomarkers for mature B cells and follicular dendritic cells (but is not limited thereto).

Multispectral immunofluorescence detection of multiple biomarkers for many different research, diagnostic, and prognostic purposes are specifically contemplated as part of the methods described herein. The inventors believe, in view of the present invention, that a skilled person using the methods of the invention described herein can simultaneously detect the presence and abundance of multiple cells or cell types as follows.

The skilled person may select a suitable set of at least three, preferably four, five, six, preferably at least seven antibodies that specifically bind to at least three, preferably four, five, six, preferably at least seven target antigens, each comprised on a predetermined biomarker, respectively. The selected antibody is then conjugated directly to one of the FPs described herein to form an Ab-FP conjugate described herein for simultaneous multispectral imaging of the biomarker.

In this manner, the skilled artisan can employ the methods described herein to conduct studies of various cells, cell populations, and tissues, and to conduct diagnoses and/or prognoses for a number of different diseases or conditions. In some embodiments, the disease or disorder is an immunological disease or disorder. The methods described herein may also be used clinically to identify patient populations and sub-populations by immune populations and sub-populations of cells, and to monitor the effect of drugs and/or drug candidates on target antigen expression, but are not so limited.

By way of non-limiting example, an immune population of cells relevant to identification in a clinical setting includes CD4+ T cells including regulatory T cells, CD8+ T cells (cytotoxic), B cells (including naive)

Activated, memory and plasma cells), monocytes, dendritic cells, macrophages and tumor cells.

The inventors believe that the advantages of the methods and reagents provided herein are numerous when compared to methods available in the art.

Traditional microscope-based immunofluorescence microscopy (IFM) is limited to the detection of four different labels, each label corresponding in color to itself correspond to the fluorescence emission spectrum of a different fluorophore included in the label. The use of nuclear stains (e.g., DAPI or HOECHST33342) means that the skilled practitioner can visualize up to three proteins and the nuclear stain simultaneously in a single field of view (e.g., via a rapid exchange filter set).

IFM in clinical pathology is usually only used for generating monochromatic images, e.g. for detecting antibody deposition in organs such as the kidney. In contrast, when visualization of biomarkers is required, clinical pathologists will typically use enzyme Immunohistochemistry (IHC). Although two or even three color methods can be used for immunohistochemical staining, most clinical pathology laboratories use only a single color, the usual visualization being based on the binding of horseradish peroxidase-labeled secondary antibodies. Quantification of cells is usually performed manually. Manual visualization can be difficult and prone to inaccuracies and errors, relying on interpretation in many cases, for example, in visualizing cancerous cells. The skilled person must rely on the size and shape of the cells to identify cancer cells and then count how many of the identified cells are labeled. This process is known to be fraught with inaccuracies.

Currently employed multiplex IFM methods generally allow the identification of up to four proteins simultaneously. Traditional multiplex IFM methods (paraffin embedded (FFPE) tissue fixed with formalin) are typically performed in two to four colors for the following reasons: 1) limited availability of primary antibodies in different isotypes (IF derived from the same host animal) and host species, 2) limited availability of fluorophores that can be separated well by conventional IF microscope filters.

Current applications using multi-spectral imaging have increased the number of colors that can be visualized, and the field of view that can be analyzed. In particular, a multispectral scan may be used to generate a multispectral image that includes a plurality of colors, each color corresponding to the fluorescence emission spectrum of a different fluorophore. Multispectral visualization of different emission spectra of the entire sample can be done in a single scan.

However, current methods for multiple labeling of tissue sections for multispectral imaging suffer from a number of recognized disadvantages, including the long time required to prepare the sample for multiple imaging, lack of reproducibility, and high cost.

For example, some iterative tagging schemes may provide a large number of different colors (>40 colors); for example, the CODEX system (https:// www.akoyabio.com/codextm/technology). The CODEX system is an IFM-based platform that uses an iterative labeling scheme with oligonucleotide-conjugated abs, added 3 times per antibody binding cycle, followed by repeated stripping and re-binding of the antibodies to generate a multi-labeled sample. There are currently only a limited number of oligonucleotide-conjugated antibodies available. These antibodies are specialized, expensive labeled antibodies, rather than the existing FP-conjugated antibodies. CODEX may be equipped with a multispectral scanner, but multispectral imaging using CODEX is limited to three colors. Due to the iterative marking process, the modification of existing marking schemes as well as the production of new schemes and marking panels for this platform is slow.

In CODEX, generation of tissue samples that allow such imaging is not accomplished by simultaneous labeling of tissue sections with the various Ab-FPs described herein. In contrast, this technique requires the tedious staining and stripping protocols mentioned above, and employs secondary fluorescent labeling via oligonucleotide tags.

In contrast, the methods described herein are clearly advantageous for clinicians in enabling equivalent staining protocols to be performed in as little as 2 hours and iterated over several days. The speed of the multispectral scan employed in the methods described herein facilitates these advantages. In some embodiments, generating a multispectral fluorescence image of the sample is done in less than 10min, less than 20min, preferably less than 30 min.

Examples of currently available multiplex immunofluorescence imaging methods include MACSima ^TM (Miltenyi Biotec) and Chip-cytometry Zellscanner one (Zellkraftwerk). However, each of these techniques has several disadvantages discussed herein.

MACSima and Zellscanner also require iterative labeling. The MACSima system allows only three antibodies at a time. The MACSima system is equipped with a conventional fluorescence microscope and does not allow multispectral imaging of the entire tissue section.

The Zellscanner system (http:// www.zellkraftwerk.com/products) allows the addition of up to 5 specific fluorophore conjugated Abs (BUV395, BUV421, FITC, PE, PerCP) per cycle and repeats the labeling cycle. However, Zellscanner is limited to using these 5 fluorophores because the instrument is specifically designed for these fluorophores alone. The Zellscanner is equipped with a conventional fluorescence microscope and cannot perform multispectral scans. Another major drawback of this system is that the tissue slices to be examined must be loaded onto the particular chip to be examined by the instrument. This requirement makes Zellscanner difficult to use with tissue samples that have been frozen or paraffin embedded.

Again, due to the iterative marking process, the modification of existing marking schemes as well as the production of new schemes and marking panels for MACSima and Zellscanner platforms is slow.

Ab-FP labeling of formalin-fixed paraffin-embedded (FFPE) tissue

Antibody labeling of FFPE tissue using traditional multiplex IF methods (i.e. indirect IF with primary antibody to target and fluorophore conjugated secondary antibody conjugated to primary antibody) is mainly performed in two to four colors due to 1) limited availability of different isotypes (IF from the same host animal) and primary antibodies in the host species, 2) limited availability of fluorophores that can be well separated by conventional IF microscope filters.

Staining FFPE tissue IF to two to four colors using traditional multiplex IFM methods typically takes one and a half to two days.

The new Opal IHC technology allows for multiplex IF staining of FFPE tissue in up to seven to nine colors (provided that a microscope capable of multispectral imaging is available). However, Opal staining requires a minimum of three to four days to complete the entire staining cycle. Furthermore, developing and optimizing a seven-color Opal staining panel may require six to eight weeks (for more information see the webpage: https:// www.akoyabio.com/product-support/optical-multiplex-immunological chemistry # Opal-FAQ), and the user needs considerable knowledge and experience of IHC technology to design and develop an Opal staining panel. Furthermore, the Opal platform is not compatible with frozen tissue sections.

Therefore, since multiple staining using Opal multiplex IHC technology is slow, technically difficult due to the large number of steps, and can be used only with a limited number of staining reagents, this technology is not widely used in clinical diagnostic and research laboratories.

In providing the methods and reagents described herein, the inventors overcome many of the disadvantages described above by directly labeling a target antigen using the directly conjugated Ab-FP conjugates described herein in a method of simultaneously labeling and detecting biomarkers.

The methods described herein are not sandwich assays and do not involve primary labeling of the target antigen with a primary antibody that specifically binds to the target antigen, followed by secondary labeling of the primary antibody with an antibody-fluorophore conjugate, followed by detection of the fluorophore by fluorescence emission. In contrast, the method entails an Ab-FP as described herein, wherein the antibody portion of the Ab-FP is a primary antibody that specifically binds the target antigen and the FP portion is directly linked to the primary antibody. It will be understood by those skilled in the art that following the methods described herein provides the advantage of direct detection of the target antigen by the Ab-FP described herein, without the need for secondary labeling as employed in sandwich assays known in the art.

The main advantage provided by the present invention is the use of antibodies directly conjugated to fluorophores (i.e., directly conjugated antibodies) to quickly allow for multi-spectral detection and imaging of the entire slide/slice. In some embodiments, advantages also include the use of multiple directly conjugated antibodies to quickly and simultaneously allow multispectral detection and imaging of the entire slide/section. In one non-limiting example, this is done using Vectra Polaris.

The inventors believe that they first provide the skilled person with the ability to rapidly generate a new diagnostic test that visualizes all clinically relevant cellular markers in a single image, enabling the quantification of relative expression in different cells. For example, following the methods described herein, multiplex fluorescence images of planar biological samples comprising at least three, up to twelve target antigens can be generated in less than 15 minutes. By these means, the inventors provide a surprising and effective technical solution that will quickly open up new opportunities in cell and tissue research as well as in clinical medicine. The ability to identify cells and/or cell populations and/or perform such diagnostic tests within hours as provided herein provides clear and unique advantages over the prior art and provides means for the skilled artisan to improve the efficiency and accuracy of research and diagnostic pathology applications in many areas.

The inventors further believe that the methods and reagents described herein provide non-obvious technical solutions that enable the development of new applications in biomedical research and medicine as described herein, as will be readily understood by the skilled person. The methods and reagents described herein also enable the development of rapid diagnostics that can be used to accelerate the selection of an optimal treatment regimen for a patient. For example, in some embodiments, the methods disclosed herein can be used by the skilled artisan for molecular and immunological analysis of cancer (including lung, breast and colorectal cancers) and disease management by selecting an appropriate plurality of biomarkers (e.g., references (Hofman, 2019), (Sood, 2006) and (Majtahed, 2011), the disclosures of which are expressly incorporated herein by reference in their entirety).

Thus, in one aspect, the invention relates to a composition comprising at least three, four, five, six, or at least seven antibody-fluorophore conjugates (Ab-FP), wherein each FP has a different fluorescence excitation and emission spectrum (Ex).

In one embodiment, each Ab is different from each other.

In one embodiment, the composition comprises at least eight, nine, ten, eleven, or twelve Ab-FPs. In one embodiment, the composition comprises six, seven or eight Ab-FPs.

In one embodiment, the composition consists essentially of at least three, four, five, six, seven, eight, nine, ten, eleven, or twelve Ab-FPs. In one embodiment, the composition consists essentially of at least three to at least seven Ab-FP species. In one embodiment, the composition consists essentially of at least six, seven, or eight Ab-FP species.

In one embodiment, the composition consists essentially of at least six Ab-FPs. In one embodiment, the composition consists essentially of at least seven Ab-FPs. In one embodiment, the composition consists essentially of at least eight Ab-FP species.

In one embodiment, the composition consists essentially of six Ab-FPs. In one embodiment, the composition consists essentially of seven Ab-FPs. In one embodiment, the composition consists essentially of eight Ab-FPs.

In one embodiment, each Ab-FP in the composition is a unique Ab-FP.

In one embodiment, the maximum excitation and emission wavelength (Ex/Em) of each FP is selected from the group consisting of 348/395, 404/448, 405/421, 405/510, 405/570, 405/603, 405/646, 405/711, 407/421, 415/500, 436/478nm, 490/515nm, 494/520nm, 495/519nm, 485/693nm, 496/578, 532/554nm, 566/610nm, 590/620nm, 650/660nm, 650/668nm, 652/704, 696/719nm, 753/785nm, 754/787nm, 755/775nm and 759/775 nm.

In one embodiment, at least one, two or three of the FPs have a maximum fluorescence emission wavelength (Em) from about 710nm to about 850 nm. In one embodiment, at least one, two or three of the FPs have an Em of about 753nm to about 759nm, preferably 753nm, 754nm, 755nm or 759 nm. In one embodiment, the Em of one of the FPs is 754 nm.

In one embodiment, FP is selected from Brilliant with Ex/Em of 348/395 ^TM Ultraviolet 395(BUV395), Brilliant with Ex/Em of 436/478nm ^TM Violet 480(BV480), Brilliant Violet 421 with Ex/Em 405/421 ^TM Brilliant with Ex/Em of 407/421 ^TM Brilliant with Violet 421(BV421), Ex/Em 405/510 ^TM Violet 510(BV510), Brilliant Violet 570 with Ex/Em 405/570 ^TM Brilliant Violet 605 with Ex/Em of 405/603 ^TM Brilliant Violet 650 with Ex/Em of 405/646 ^TM Brilliant Violet 711 with Ex/Em of 405/711 ^TM BD Horizon with Ex/Em of 404/448 ^TM BD Horizon with V450 and Ex/Em of 415/500 ^TM Brilliant with V500 and Ex/Em of 490/515nm ^TM Blue 515(BB515), Fluorescein Isothiocyanate (FITC) having an Ex/Em of 494/520nm, Alexa Fluor 488(AF488) having an Ex/Em of 495/519nm, Alexa Fluor 532(AF532) having an Ex/Em of 532/554nm, a salt thereof, and a salt thereof,R-Phycoerythrin (PE) with Ex/Em of 496/578, Alexa Fluor 594(AF594) with Ex/Em of 590/620nm, PE-Dazle 594(PE594) or PE-CF594(CF594) with Ex/Em of 566/610nm, Alexa Fluor 647(AF647) with Ex/Em of 650/668nm, Allophycocyanin (APC) with Ex/Em of 650/660nm, BD Horizon with Ex/Em of 485/693nm ^TM 700(BB700), Alexa Fluor 700(AF700) with Ex/Em of 696/719nm, APC/Alexa Fluor 750 with Ex/Em of 753/785nm, APC/Fire 750 with Ex/Em of 754/787nm, APC-R700 with Ex/Em of 652/704, APC-Cy7 with Ex/Em of 755/775nm and AF750 with Ex/Em of 759/775 nm.

In one embodiment, the fluorophore is selected from the group consisting of BV480, BB515, AF532, PE-CF594, AF647, and AF700 or BB 700.

In one embodiment, the fluorophore is selected from the group consisting of BV480, BB515, AF532, PE-CF594, AF647, AF700, and BB 700.

In one embodiment, the fluorophore is selected from the group consisting of AF647, AF488, AF594, AF700, BV510, BV650, PE/Dazle 594, and APC/Fire 750.

In one embodiment, the Ab in Ab-FP is selected from the group consisting of anti-CD 31, CD141, CD144, CD3, CD34, CD163, CD11c, CD14, CD16, CD68Foxp3, CD4, CD8, CD19, CD20, CD25, CD45RO, CD45RA, CD38, PD-1, PDL1, PDL2, CD68, Ki-67, Sox10, S100, PRAME, MART1, and anti-CD 21 antibodies.

In one embodiment, Ab in Ab-FP is selected from the group consisting of CD19, CD8, and CD45 RO.

In one embodiment, the abs in Ab-FP include one or more abs selected from the group consisting of anti-Estrogen Receptor (ER), Progesterone Receptor (PR), her2, and anti-cytokeratin antibodies.

In one embodiment, the abs in Ab-FP include one or more anti-mismatch repair protein antibodies or anti-mutant mismatch repair protein antibodies.

In one embodiment, the Ab in Ab-FP comprises one or more antibodies selected from the group consisting of anti-b-raf (V600E mutation), MLH1, MSH2, MSH6, and anti-PMS 2 antibodies.

In one embodiment, the Ab in Ab-FP is selected from the group consisting of anti-CD 31, CD141, CD3, CD34, Ki-67, CD11c, and anti-CD 21 antibodies.

In one embodiment, the composition comprises at least three, preferably four, five or preferably all six of the following antibodies: anti-CD 31, CD141, CD3, CD34, Ki-67, and anti-CD 21 antibodies.

In one embodiment, the composition comprises three, preferably four, five or preferably all six of the following antibodies: anti-CD 31, CD141, CD3, CD34, Ki-67, and anti-CD 21 antibodies.

In one embodiment, the composition comprises at least one, preferably at least two of the following antibodies: anti-CD 19, CD8, and anti-CD 45RO antibodies.

In one embodiment, the composition comprises one, preferably two, of the following antibodies: anti-CD 19, CD8, and anti-CD 45RO antibodies.

In one embodiment, the composition comprises at least three, preferably four, five or preferably all six of the following Ab-FPs: CD31-BV 480; CD141-BB 515; CD3-AF 532; CD34-PE-CF 594; ki67-AF647 and CD21-BB 700.

In one embodiment, the composition comprises three, preferably four, five or preferably all six of the following Ab-FPs: CD31-BV 480; CD141-BB 515; CD3-AF 532; CD34-PE-CF 594; ki67-AF647 and CD21-BB 700.

In one embodiment, the composition comprises at least three, preferably four, five, six or preferably all seven of the following antibodies: anti-CD 31, CD141, CD3, CD34, Ki-67, CD11c, and anti-CD 21 antibodies.

In one embodiment, the composition comprises three, preferably four, five, six or preferably all seven of the following antibodies: anti-CD 31, CD141, CD3, CD34, Ki-67, CD11c, and anti-CD 21 antibodies.

In one embodiment, the composition comprises at least three, preferably four, five, six or preferably all seven of the following Ab-FPs: CD31-BV 480; CD141-BB 515; CD3-AF 532; CD34-PE-CF 594; ki67-AF647, CD11c-AF700 and CD21-BB 700.

In one embodiment, the composition comprises three, preferably four, five, six or preferably all seven of the following Ab-FPs: CD31-BV 480; CD141-BB 515; CD3-AF 532; CD34-PE-CF 594; ki67-AF647, CD11c-AF700 and CD21-BB 700.

In one embodiment, the composition comprises at least one, preferably at least two, of the following Ab-FPs: CD19-AF 647; CD8-AF 647; CD45RO-AF 488; CD45RO-AF 594; CD45RO-AF 700; CD45RO-BV 510; CD45RO-BV 650; CD45 RO-PE/Dazle 594 and CD45RO-APC/Fire 750.

In one embodiment, the composition comprises one, preferably two, of the following Ab-FPs: CD19-AF 647; CD8-AF 647; CD45RO-AF 488; CD45RO-AF 594; CD45RO-AF 700; CD45RO-BV 510; CD45RO-BV 650; CD45RO-PE/Dazzle594 and CD45RO-APC/Fire 750.

In one embodiment, each Ab specifically binds to a target antigen. In one embodiment, each Ab specifically binds a different target antigen. In one embodiment, each target antigen is a biomarker for a protein or cell type. In one embodiment, each target antigen is a biomarker for a different protein or different cell type.

In one embodiment, each target antigen is a biomarker for a cell surface receptor or an intracellular antigen. In one embodiment, the intracellular antigen is a nuclear antigen.

In one embodiment, each target antigen is a T cell, B cell, macrophage, monocyte, or dendritic cell antigen.

In one embodiment, each target antigen is a T cell antigen selected from the group consisting of CD3, CD4, CD8, FoxP3, CD25, CD137, CD38, CD69, PD-1, CTLA-4, CD45RO, CD45RA, and Ki67 antigens.

In one embodiment, each target antigen is a B cell antigen selected from the group consisting of CD19, CD20, CD21, BCL-6, blip 1, Ki67, and CD138 antigens.

In one embodiment, each target antigen is a macrophage or monocyte antigen selected from the group consisting of CD14, CD16, CD68, and CD163 antigens.

In one embodiment, each target antigen is a dendritic cell antigen, i.e., CD1c or CLEC9a antigen.

In one embodiment, the target antigen is a biomarker associated with a disease or disorder.

In one embodiment, the biomarker is a diagnosis of a disease or disorder.

In one embodiment, the biomarker is a prognosis of the disease or disorder. In one embodiment, the disease or disorder is cancer, preferably breast cancer, lung cancer, colorectal cancer or melanoma. In one embodiment, the disease or disorder is an immunological disease or disorder.

a) Labeling at least one target antigen in a planar biological sample with at least one unique Ab-FP conjugate, and

c) determining from the image the presence or absence of at least one biomarker comprising at least one target antigen.

In one embodiment, labeling in a) comprises simultaneously labeling at least two, preferably at least three, four, five, six, preferably at least seven different target antigens with at least two, preferably at least three, four, five, six, preferably at least seven unique Ab-FP conjugates.

In one embodiment, labeling in a) comprises simultaneously labeling two, preferably three, four, five, six, preferably seven different target antigens with two, preferably three, four, five, six, preferably seven unique Ab-FP conjugates.

In one embodiment, one color in b) is correlated with the fluorescence emission spectrum of the nuclear dye. In one embodiment, the nuclear stain is DAPI or Hoechst 33342.

In one embodiment, the multispectral image in b) comprises at least three, preferably at least four, five, six, preferably seven different colors, wherein each different color is associated with specific binding of Ab-FP and the target antigen.

In one embodiment, the multispectral image in b) comprises three, preferably at least four, five, six, preferably seven different colors, wherein each different color is associated with specific binding of Ab-FP and the target antigen.

In one embodiment, generating the multispectral image in b) comprises separately detecting at least two, preferably three, four, five, six, preferably seven different fluorescence spectra using a multispectral scanner, wherein each detected spectrum corresponds to an Ab-FP that specifically binds to the target antigen.

In one embodiment, generating the multispectral image in b) comprises detecting two, preferably three, four, five, six, preferably seven different fluorescence spectra separately using a multispectral scanner, wherein each detected spectrum corresponds to an Ab-FP that specifically binds to the target antigen.

In one embodiment, the planar biological sample is a tissue section. In one embodiment, the tissue section is a frozen or formalin fixed tissue section. In one embodiment, the tissue section is a frozen tissue section. In one embodiment, the tissue section is a formalin-fixed paraffin-embedded tissue section.

In one embodiment, the tissue section is from a mammal, preferably a human.

In one embodiment, the tissue section is from the liver, lung, breast, colon, tonsil, or lymph node. In one embodiment, the tissue section is from a biopsy of tissue having or suspected of having at least one cancer cell. In one embodiment, the tissue biopsy is from the liver, lung, breast, colon, or lymph node. In one embodiment, the tissue biopsy is a cancer biopsy. In one embodiment, the tissue biopsy is a tumor biopsy.

In one embodiment, the at least one target antigen is a biomarker for a biomolecule. In one embodiment, the biomolecule is or includes a protein, carbohydrate or lipid or antigenic portion thereof. In one embodiment, the biomolecule is a protein or antigenic portion thereof. In one embodiment, the protein is expressed in or on a cell, cellular structure or cellular substructure.

In one embodiment, the at least one target antigen is a biomarker for a cell, cell type, cell structure, or cell substructure. In one embodiment, the presence of a biomarker defines a cell type. In one embodiment, the presence of one or more biomarkers defines a cell type.

In one embodiment, the abundance of the biomarker defines the cell type. In one embodiment, the abundance of one or more biomarkers defines the cell type. In one embodiment, the relative abundance of the biomarkers defines the cell type. In one embodiment, the relative abundance of one or more biomarkers defines the cell type.

In one embodiment, the biomolecule is a biomarker for a cell surface receptor or an intracellular antigen. In one embodiment, the intracellular antigen is a nuclear antigen.

In one embodiment, the at least one target antigen is a biomarker for T cells, B cells, macrophages, monocytes or dendritic cells.

In one embodiment, the at least one target antigen is selected from the group consisting of anti-Estrogen Receptor (ER), Progesterone Receptor (PR), her2, and anti-cytokeratin antigen.

In one embodiment, the at least one target antigen is a mismatch repair protein or a mutant mismatch repair protein antigen.

In one embodiment, the at least one target antigen is selected from the group consisting of b-raf (V600E mutation), MLH1, MSH2, MSH6 and PMS2 antigens.

In one embodiment, the at least one target antigen is a T cell antigen selected from the group consisting of CD3, CD4, CD8, FoxP3, CD25, CD137, CD38, CD69, PD-1, CTLA-4, CD45RO, CD45RA, and Ki67 antigens.

In one embodiment, the at least one target antigen is a B cell antigen selected from the group consisting of CD19, CD20, CD21, BCL-6, blip 1, Ki-67, and CD138B cell antigens.

In one embodiment, the at least one target antigen is the dendritic cell antigen CD1c or CLEC9a antigen.

In one embodiment, the at least one target antigen is a biomarker associated with a disease or disorder. In one embodiment, the biomarker is a diagnosis or partial diagnosis of a disease or disorder. In one embodiment, the biomarker is a prognosis or partial prognosis of the disease or disorder. In one embodiment, the disease or disorder is cancer, preferably breast, lung or colorectal cancer or melanoma.

In one embodiment, the biomarkers distinguish or partially distinguish a patient population.

In one embodiment, the biomarker distinguishes, or partially distinguishes, patients who respond to a particular therapy from patients who do not respond.

In one embodiment, the biomarker distinguishes, or partially distinguishes, patients more likely to respond to a particular therapy from patients less likely to respond.

In one embodiment, the therapy is immunotherapy. In one embodiment, the therapy is a cancer therapy.

In one embodiment, the Ab in Ab-FP is selected from the group consisting of anti-CD 31, CD141, CD3, CD34, CD163, CD11c, CD14, CD16, Foxp3, CD4, CD8, CD19, CD20, CD25, CD38, CD45RO, CD45RA, PD-1, PDL1, PDL2, CD68, CD14, Ki-67, Sox10, S100, PRAME, MART1, and anti-CD 21 antibodies.

In one embodiment, the Ab in Ab-FP comprises one or more antibodies selected from the group consisting of anti-Estrogen Receptor (ER), Progesterone Receptor (PR), her2, and anti-cytokeratin antibodies.

In one embodiment, the abs in Ab-FP include one or more anti-mismatch repair protein or mutant mismatch repair protein antibodies.

In one embodiment, the Ab in Ab-FP is selected from the group consisting of CD19, CD8, and CD45 RO.

In one embodiment, the labeling in a) comprises simultaneous labeling with two to seven unique Ab-FPs, preferably with six unique Ab-FPs, preferably with seven unique Ab-FPs.

In one embodiment, the labeling in a) further comprises simultaneous labeling with at least two Ab-FPs, preferably with at least three, four, five or six additional unique Ab-FPs.

In one embodiment, the labeling in a) comprises labeling with at least two, preferably three, four, five, six or preferably all seven of the following antibodies: anti-CD 31, CD141, CD3, CD34, Ki67, CD11c and anti-CD 21 antibodies.

In one embodiment, the labeling in a) comprises labeling with two, preferably three, four, five, six or preferably all seven of the following antibodies: anti-CD 31, CD141, CD3, CD34, Ki67, CD11c and anti-CD 21 antibodies.

In one embodiment, the labeling in a) comprises labeling with at least two, preferably three, four, five, six, or preferably all seven of the following Ab-FP: CD31-BV 480; CD141-BB 515; CD3-AF 532; CD34-PE-CF 594; ki67-AF 647; CD11c-AF700 and CD21-BB 700.

In one embodiment, the labeling in a) comprises labeling with two, preferably three, four, five, six, or preferably all seven of the following Ab-FP: CD31-BV 480; CD141-BB 515; CD3-AF 532; CD34-PE-CF 594; ki67-AF 647; CD11c-AF700 and CD21-BB 700.

In one embodiment, the labeling in a) comprises labeling with at least one, preferably at least two of the following antibodies: anti-CD 19, CD8, and anti-CD 45RO antibodies.

In one embodiment, the labeling in a) comprises labeling with one, preferably two, of the following antibodies: anti-CD 19, CD8, and anti-CD 45RO antibodies.

In one embodiment, the labeling in a) comprises labeling with at least one, preferably at least two of the following Ab-FP: CD19-AF 647; CD8-AF 647; CD45RO-AF 488; CD45RO-AF 594; CD45RO-AF 700; CD45RO-BV 510; CD45RO-BV 650; CD45 RO-PE/Dazle 594 and CD45RO-APC/Fire 750.

In one embodiment, the labeling in a) comprises labeling with one, preferably two, of the following Ab-FP: CD19-AF 647; CD8-AF 647; CD45RO-AF 488; CD45RO-AF 594; CD45RO-AF 700; CD45RO-BV 510; CD45RO-BV 650; CD45 RO-PE/Dazle 594 and CD45RO-APC/Fire 750.

In one embodiment, the labeling in a) further comprises replacing one Ab-FP with a different Ab-FP. In one embodiment, the replacement Ab-FP includes the same Ab and an FP having an Ex/Em of about 753nm to about 759nm, preferably 753nm, 754nm, 755nm, or 759 nm.

In one embodiment, labeling in a) further comprises labeling an additional target antigen with an additional Ab-FP comprising an FP having an Ex/Em of about 753nm to about 759nm, preferably 753nm, 754nm, 755nm, or 759 nm.

In one embodiment, the additional Ab-FP includes an anti-CD 31, CD141, CD3, CD34, Ki-67, CD11c, or anti-CD 21 Ab. In one embodiment, the additional Ab does not include an anti-CD 31, CD141, CD3, CD34, Ki-67, CD11c, or anti-CD 21 Ab.

In one embodiment, the determination in c) is the detection of the presence of at least two, preferably at least three, four, five, six, preferably at least seven different biomarkers.

In one embodiment, the determination in c) is the detection of the presence of two, preferably at least three, four, five, six, preferably at least seven different biomarkers.

In one embodiment, the determination in c) comprises determining the abundance of at least two, preferably at least three, four, five, six, preferably at least seven different biomarkers. In one embodiment, determining the abundance is determining the relative abundance of each of the different biomarkers.

In one embodiment, the determination in c) comprises determining the abundance of two, preferably at least three, four, five, six, preferably at least seven different biomarkers. In one embodiment, determining the abundance is determining the relative abundance of each of the different biomarkers.

In one embodiment, determining the abundance determined in c) comprises determining the number of cells of the one or more labeled biomarkers in the image.

In one embodiment, determining the relative abundance comprises determining in c) the percentage of cells of a predetermined cell type in the image that comprises one or more labeled biomarkers within the total number of cells of the cell type observable in the image. In one embodiment, the cells of the cell type are determined by the presence of one or more biomarkers. In one embodiment, the cells of the cell type are determined by morphological criteria. In one embodiment, the cells of the cell type are determined by a combination of the presence of one or more biomarkers and morphological criteria.

In one embodiment, generating the image in b) comprises subtracting a fluorescence emission spectrum corresponding to background autofluorescence from the image.

In one embodiment, the one or more biomarkers determined in c) are high abundance in individuals with a predetermined disease or condition. In one embodiment, the one or more biomarkers determined in c) are relatively abundant in individuals with a predetermined disease or disorder.

In one embodiment, the predetermined disease or condition is a known disease or condition.

In one embodiment, the predetermined disease or condition is one for which detection methods are currently available but are not clinically applicable.

In one embodiment, the predetermined cell type is a cell type known to be associated with a disease or disorder.

In one embodiment, the determination in c) comprises detecting one or more biomarkers on or in cells of the immune system.

In one embodiment, the determination in c) comprises detecting one or more biomarkers of a defined cell type, preferably an immune cell type.

In one embodiment, the determination in c) comprises determining that the one or more biomarkers are present in or on a cell of the immune system in high abundance or relatively high abundance.

In one embodiment, the cells of the immune system are selected from the group consisting of T cells, B cells, macrophages, monocytes and dendritic cells.

In one embodiment, the determination in c) comprises detecting one or more biomarkers on or in at least one tumor cell.

In one embodiment, the determining in c) comprises detecting a clinically relevant level of one or more biomarkers.

In one embodiment, the determining in c) comprises determining that the one or more biomarkers are present in high abundance or relatively high abundance in a sample from a subject having a predetermined disease or disorder. In one embodiment, the predetermined disease or disorder is an immunological disease or disorder. In one embodiment, the predetermined disease or disorder is cancer, preferably lung cancer, breast cancer or colorectal cancer and/or melanoma.

In one embodiment, the one or more biomarkers are present in a relatively high abundance in a sample from an individual suspected of having a predetermined disease or disorder. In one embodiment, the predetermined disease or disorder is an immunological disease or disorder.

In one embodiment, the multispectral image in b) is generated by computer image analysis of fluorescence intensity data from each pixel in the image captured by the multispectral scanner.

In one embodiment, the multispectral scanner is Vectra Polaris.

In one embodiment, the data generated by the multispectral scanner is image analyzed using InForm cell analysis software or Halo software (PerkinElmer) to generate the image in b).

In one embodiment, the image generated in b) comprises at least two distinct colors, preferably at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, preferably twelve distinct colors, wherein each color is associated with a different FP fluorescence emission spectrum.

In one embodiment, the image generated in b) comprises two distinct colors, preferably three, four, five, six, seven, eight, nine, ten, eleven, preferably twelve distinct colors, wherein each color is associated with a different FP fluorescence emission spectrum.

In one embodiment, the image generated in b) comprises at least four, preferably at least five, six, seven or eight unique colors, wherein each color is associated with a different FP fluorescence emission spectrum.

In one embodiment, the image generated in b) comprises four, preferably five, six, seven or eight unique colors, wherein each color is associated with a different FP fluorescence emission spectrum.

In one embodiment, the image generated in b) comprises at least six, seven or eight distinct colors, wherein each color is associated with a different FP fluorescence emission spectrum.

In one embodiment, the image generated in b) comprises six, seven or eight unique colors, wherein each color is associated with a different FP fluorescence emission spectrum.

In one embodiment, the image generated in b) comprises six distinct colors, wherein each color is associated with a different FP fluorescence emission spectrum.

In one embodiment, the image generated in b) comprises seven distinct colors, wherein each color is associated with a different FP fluorescence emission spectrum.

In one embodiment, the image generated in b) comprises eight distinct colors, wherein each color is associated with a different FP fluorescence emission spectrum.

In one embodiment, the at least one, two, or three unique colors specifically correspond to FP emission spectra of about 710nm and about 850 nm. In one embodiment, the FP maximum emission wavelength for the at least one unique color specific corresponds to about 753nm to about 759nm, preferably 753nm, 754nm, 755nm, or 759 nm.

In one embodiment, one, two, or three unique colors specifically correspond to FP emission spectra of about 710nm and about 850 nm. In one embodiment, a unique color specifically corresponds to a FP having a maximum emission wavelength of about 753nm to about 759nm, preferably 753nm, 754nm, 755nm, or 759 nm.

In one embodiment, the multispectral immunofluorescence image of the planar biological sample comprises three, preferably four, five, six, seven, preferably eight colors, wherein three, preferably four, five, six, preferably seven colors are associated with specific binding of three, preferably four, five, six, preferably seven Ab-FP and the target antigen comprised in the planar sample.

In one embodiment, a multispectral image is generated according to the methods described herein.

In one embodiment, the multispectral image is generated by computer image analysis of fluorescence intensity data from each pixel in an image captured by the multispectral scanner according to the methods described herein.

In one embodiment, the multispectral scanner is Vectra Polaris.

As embodiments of this aspect of the invention relating to multispectral immunofluorescence images, specifically contemplated are all embodiments set forth in previous aspects of the invention herein, particularly and not limited to embodiments relating to antibodies, fluorophores, Ab-FP, target antigens, biomarkers, tissues, cells, cell types, cellular structures and substructures, markers, and multispectral imaging, but not limited thereto.

In one embodiment, the target antigens are different from each other.

In one embodiment, the at least two target antigens are present on or in different cell types in the sample.

In one embodiment, the multispectral image in b) comprises at least three, preferably at least four, five, six, seven, preferably eight different colors, wherein at least two, preferably three, four, five, six, preferably seven different colors are associated with specific binding of Ab-FP and target antigen.

In one embodiment, the multispectral image in b) comprises three, preferably four, five, six, seven, preferably eight different colors, wherein two, preferably three, four, five, six, preferably seven different colors are associated with specific binding of Ab-FP and target antigen.

In one embodiment, generating the multispectral image in b) comprises separately detecting at least two, preferably three, four, five, six, preferably seven different fluorescence spectra using a multispectral scanner, each detected spectrum corresponding to an Ab-FP that specifically binds to the target antigen.

In one embodiment, generating the multispectral image in b) comprises detecting two, preferably three, four, five, six, preferably seven different fluorescence spectra separately using a multispectral scanner, each detected spectrum corresponding to an Ab-FP that specifically binds to the target antigen.

In one embodiment, the labeling in a) comprises simultaneous labeling with at least three, preferably with at least four, five, six, seven, eight, nine, eleven, or preferably at least twelve unique Ab-FPs.

In one embodiment, the labeling in a) comprises simultaneous labeling with three Ab-FPs, preferably with four, five, six, seven, eight, nine, eleven, or preferably at least twelve unique Ab-FPs.

In one embodiment, the labeling in a) comprises simultaneous labeling with three to seven unique Ab-FPs, preferably six unique Ab-FPs, preferably seven unique Ab-FPs. In one embodiment, the planar biological sample is a tissue section. In one embodiment, the tissue section is a frozen or formalin fixed tissue section. In one embodiment, the tissue section is a frozen tissue section. In one embodiment, the tissue section is a formalin-fixed paraffin-embedded tissue section.

Specifically contemplated herein as embodiments of this aspect of the invention of the method of detecting multiple target antigens are all embodiments set forth herein relating to the previous aspects of the invention set forth herein, including but not limited to antibodies, fluorophores, Ab-FP, target antigens, biomarkers, tissues, cell types, markers, multispectral imaging, and multispectral image analysis.

In one embodiment, the determination in c) comprises determining the presence or abundance of a cell type based on labeling the cell type with a unique Ab-FP.

Specifically contemplated herein as embodiments of this aspect of the invention of the method of detecting a plurality of biomarkers are all embodiments set forth herein relating to the previous aspects of the invention set forth herein, including but not limited to antibodies, fluorophores, Ab-FP, target antigens, biomarkers, tissues, cell types, and methods of detecting a plurality of target antigens, including but not limited to labeling, multispectral imaging, and multispectral image analysis.

b) generating a multispectral fluorescence image of the labeled planar sample using a multispectral scanner, wherein the image comprises at least three colors, wherein at least two colors are associated with specific binding of each unique Ab-FP conjugate and a target antigen on different cell types, and

In one embodiment, the cell type is defined by the presence of at least one, preferably two, three, four, five, six or at least seven different biomarkers.

In one embodiment, the cell type is defined by the presence of one, preferably two, three, four, five, six or at least seven different biomarkers.

As embodiments of this aspect of the invention of the method of detecting a plurality of different cell types in a biological sample, specifically contemplated herein are all embodiments set forth herein relating to the previous aspects of the invention, including but not limited to antibodies, fluorophores, Ab-FP, target antigens, biomarkers, tissues, cell types, and methods of detecting target antigens, biomarkers, and cell types, including but not limited to labeling, multispectral imaging, and multispectral image analysis.

c) based on the detected fluorescence emission spectra, optionally according to suitable reference controls, the abundance of a plurality of different cell types in the sample is determined.

In one embodiment, the determination in c) comprises subtracting a fluorescence emission spectrum corresponding to background autofluorescence from the image generated in b).

In one embodiment, the abundance is a relative abundance.

In one embodiment, the relative abundance is the percentage of cells in the total cell population determined to be one cell type.

In one embodiment, simultaneous detection is accomplished using a multispectral scanner, preferably Vectra Polaris.

As embodiments of this aspect of the invention of the method of identifying the abundance of a plurality of different cell types in a biological sample, specifically contemplated herein are all embodiments set forth herein relating to the previous aspects of the invention set forth herein, including but not limited to antibodies, fluorophores, Ab-FP, target antigens, biomarkers, tissues, cell types, and methods of detecting a plurality of target antigens, biomarkers, and cell types, including but not limited to labeling, multispectral imaging, and multispectral image analysis.

In one embodiment, the determining in d) comprises determining the likelihood that at least one labeled cell type in the image generated in b) is within n cells of one or more different labeled cell types in the image, wherein n ═ is selected from an integer of one to ten.

As embodiments of this aspect of the invention of the method of determining the spatial distribution of a plurality of cell types in a biological sample, specifically contemplated herein are all embodiments set forth herein relating to the previous aspects of the invention set forth herein, including but not limited to antibodies, fluorophores, Ab-FP, target antigens, biomarkers, tissues, cell types, and methods of detecting a plurality of target antigens, biomarkers, and cell types, including but not limited to labeling, multispectral imaging, and multispectral image analysis.

c) detecting the presence or abundance of each biomarker in the image generated in b), wherein each biomarker is identified in the image as a different color associated with specific binding of a unique Ab-FP, an

In one embodiment, each biomarker is present on a different cell type in the sample.

In one embodiment, the subset of patients is a group of patients having a common cellular phenotype.

In one embodiment, a subgroup of patients is defined by the presence of one or more target antigens, biomarkers, and/or cell types.

As embodiments of this aspect of the invention of a method of identifying a subset of patients from a group of patients, specifically contemplated herein are all embodiments set forth herein relating to the previous aspects of the invention set forth herein, including but not limited to antibodies, fluorophores, Ab-FP, target antigens, biomarkers, tissues, cell types, and methods of detecting a variety of target antigens, biomarkers, and cell types, including but not limited to labeling, multispectral imaging, and multispectral image analysis.

f) selecting a unique Ab-FP conjugate that can be identified in the image in e) as a set of Ab-FPs for diagnosing the predetermined disease or condition in a).

Embodiments of this aspect of the invention as a method of making a diagnostic panel of antibody-fluorophore conjugates are specifically contemplated herein all embodiments set forth herein relating to previous aspects of the invention set forth herein, including but not limited to antibodies, fluorophores, Ab-FP, target antigens, biomarkers, tissues, cell types, and methods of detecting a variety of target antigens, biomarkers, and cell types, including but not limited to labeling, multispectral imaging, and multispectral image analysis.

a) generating a first multispectral immunofluorescence image from the single planar biological sample using a first set of at least two to at least seven unique Ab-FP conjugates according to the methods described herein, the first multispectral immunofluorescence image comprising up to eight different colors, wherein up to seven colors are each associated with a unique Ab-FP conjugate,

c) identification of suitable antibodies for replacement of Ab-FP

d) Identification of suitable fluorophores for replacement of Ab-FP

e) The replacement Ab-FP was obtained,

As embodiments of this aspect of the invention for identifying alternative antibody-fluorophore (Ab-FP) conjugates for use in direct immunofluorescence analysis of biological samples, specifically contemplated herein are all embodiments set forth herein that relate to previous aspects of the invention set forth herein, including but not limited to antibodies, fluorophores, Ab-FP, target antigens, biomarkers, tissues, cell types, and methods of detecting a variety of target antigens, biomarkers, and cell types, including but not limited to labeling, multispectral imaging, and multispectral image analysis.

As embodiments of this aspect of the invention of the method for determining whether at least one cell type is responsive to a drug candidate, specifically contemplated herein are all embodiments set forth herein relating to the previous aspects of the invention set forth herein, including but not limited to antibodies, fluorophores, Ab-FP, target antigens, biomarkers, tissues, cell types, and methods of detecting a variety of target antigens, biomarkers, and cell types, including but not limited to labeling, multispectral imaging, and multispectral image analysis.

In another aspect, the invention relates to a method for predicting a patient's treatment response to a proposed treatment for a predetermined disease or condition, the method comprising:

As embodiments of this aspect of the invention for predicting a therapeutic response in a patient, specifically contemplated herein are all embodiments set forth herein relating to the previous aspects of the invention set forth herein, including but not limited to antibodies, fluorophores, Ab-FP, target antigens, biomarkers, tissues, cell types, and methods of detecting a variety of target antigens, biomarkers, and cell types, including but not limited to labeling, multispectral imaging, and multispectral image analysis.

In another aspect, the invention relates to a method of identifying a cellular response to a drug candidate comprising

As embodiments of this aspect of the invention of the method of identifying a cellular response to a drug candidate, specifically contemplated herein are all embodiments set forth herein relating to the previous aspects of the invention set forth herein, including but not limited to antibodies, fluorophores, Ab-FP, target antigens, biomarkers, tissues, cell types, and methods of detecting a variety of target antigens, biomarkers, and cell types, including but not limited to labeling, multispectral imaging, and multispectral image analysis.

e) deriving an overall score for the patient from the distribution function;

g) Optionally treating the subject with a specified therapy.

As embodiments of this aspect of the invention of the method of identifying a patient who will benefit from a candidate therapy, specifically contemplated herein are all embodiments set forth herein relating to the previous aspects of the invention set forth herein, including but not limited to antibodies, fluorophores, Ab-FP, target antigens, biomarkers, tissues, cell types, and methods of detecting a variety of target antigens, biomarkers, and cell types, including but not limited to labeling, multispectral imaging, and multispectral image analysis.

In this specification, reference has been made to patent specifications, other external documents, or other sources of information, which are generally intended to provide a context for discussing the features of the invention. Citation of such external documents shall not be construed as an admission that such documents are available unless otherwise specifically indicated; or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

The invention will now be illustrated in a non-limiting manner with reference to the following examples.

Examples

Materials and methods

Reagent

Fluorophore conjugated antibodies listed below were purchased from Biolegend and BD Biosciences (USA):

CD31 BV480

CD141 BB515

CD3 AF532

CD34 PE-CF594

Ki67-AF647

CD21 BB700

standard materials used in the following examples:

TissueTek OCT frozen section embedding medium (TissueTek OCT compound)

Low-temperature die

Isopentane

Liquid nitrogen

Positively charged glass slide (for tissue slice)

Ice-cold acetone

DAKO PAP pen

1X TBS

Constant humidity box

A sealing agent: 10% human serum prepared in 1 × TBS

Antibody dilution buffer: 10% human serum prepared in 1 × TBS

DAPI (used at 1: 2000 final dilution)

Prolonggold sealant

Cover glass

Tissue sample

Melanoma infiltrated lymph nodes were provided by our clinical co-workers from the hospital of auckland city.

Slicing scheme

(1) The tissue was cut into pieces of appropriate size without rnase. If covered in blood or other liquid, the tissue is blotted on sterile gauze.

(2) A small amount of OCT cryosection embedding medium (OCT compound) was placed in the recess of the cryo-mold. Without overfilling. The tissue was centered on the bottom of the cryogenic mold. An aluminum foil mold is used if the tissue is too large to fit into the cryogenic mold.

(3) The mold was slowly filled with Tissue-Tek OCT cryosection embedding medium (Tissue-Tek OCT Compound) to avoid bubble formation until the OCT covered the Tissue.

(4) About 60 ml of isopentane was poured into a small plastic beaker. This should be sufficient to cover the tissue mass at least twice.

(5) The plastic beaker filled with isopentane was lowered into a flask containing liquid nitrogen. One third of the bottom of the beaker should be located below the surface of the liquid nitrogen. Isopentane was stirred occasionally with a spatula (5 seconds per 30 seconds). After about 4 min, small white crystals started to form at the bottom of the beaker. The isopentane was cooled sufficiently for use. If liquid nitrogen is not available, the beaker can be placed on dry ice, but isopentane will take longer to cool.

(6) The tissue/OCT filled mold was lowered with forceps into isopentane and held there until the OCT was completely frozen (about 7-10 seconds). The mold was removed and placed in a pre-cooled storage bottle.

(7) The tissue molds were stored at-80 ℃ until sectioning.

(8) OCT-embedded tissue cut to a thickness of 5 μm using a cryostat (Leica)

Labelling schemes

(1) Removing the glass slide with the frozen section from the-80 deg.C refrigerator, warming the glass slide to room temperature and drying

(2) Drawing slides with a DAKO PAP pen

(3) Tissue sections were fixed with ice-cold acetone for 5min at room temperature. The acetone should be evaporated and the slide dried.

(4) Simple washing of slides with TBS

(5) Incubating the tissue sections with the blocking agent in a dark incubator at room temperature for 10min

(6) Brushing off sealant

(7) Fluorophore conjugated antibodies prepared in antibody dilution buffer were added simultaneously to the tissue sections and incubated for 1 hour at room temperature in a dark incubator (protected from light).

(8) Briefly washed 1 time with TBS, followed by 3 times 5min each with TBS on a shaker

(9) DAPI (1: 2000) was added to the tissue sections and incubated for 5min at room temperature in a dark incubator (protected from light).

(10) Wash 3 times, 2min each time, wash with TBS on a shaker and seal slides with coverslips using prolong gold.

(11) Imaging was continued using Vectra Polaris.

Scanning protocol (from Vectra Polaris instruction 1.0.7)

(1) Opening Vectra Polaris Instrument and computer

(2) Launching Vectra Polaris software

(3) Loading slides into the stage

(4) Loading the stage into a stage Placement Chamber (slide Carrier hotel) for scanning of a microscope slide

(5) In the "edit plan" page (Vectra Polaris software), an imaging plan is created. The fluorescence mode and spatial resolution (typically x20 magnification, x10 or x20 are also available) were chosen for multi-spectral imaging (MSI) of the entire slide scan (WSS) and region of interest (ROI). The exposure times for the WSS and MSI and the filters for focusing and imaging are also set.

(6) In the "scan slide" page (Vectra Polaris software), the slide to be scanned is located and the entire slide scan (WSS) is performed using the WSS protocol created in (5)

(7) The Phenochart program (PerkinElmer) was started to view WSS images and ROI was selected for multi-spectral imaging (MSI).

(8) In the "scan slide" page (Vectra Polaris software), the slide containing the selected ROI to be imaged in a multispectral manner is located. Multispectral imaging (MSI) of selected ROI using the MSI scheme created in (5)

(9) After imaging the selected ROI, the acquired MSI images were mixed in the InForm software (PerkinElmer) using a spectral library constructed from images of singly stained tissues of each Ab-FP. Processing and analyzing the deblended image in InForm software

Example 1:

the following protocol was used to specifically detect six different target antigens in frozen sections of tumor tissue using the Ab-FP described herein.

Following the inventive method described herein, seven color multiplex immunofluorescence images (6 Ab + DAPI) were generated.

Thus, following the methods described herein allows for rapid and specific multiplexed detection of multiple target antigens without the need for a secondary antibody incubation step.

Six antibodies were added simultaneously in this protocol, while one antibody was added at a time in the seven-color opal protocol (FFPE). This example of the method of the invention thus illustrates the significant advantage provided that the total time required to specifically detect and identify multiple target antigens is greatly reduced.

This embodiment of the method of the invention uses frozen tissue and therefore does not require deparaffinization and antigen retrieval.

Material

Frozen tissue slide

Ice cold acetone

DAKO PAP pen

1X TBS

Constant humidity box

A sealing agent: 10% human serum prepared in 1 × TBS

Dilution buffer: 10% human serum prepared in 1 × TBS

Fluorophore conjugated antibodies

-CD31 BV480

-CD141 BB515

-CD3 AF532

-CD34 PE-CF594

-Ki67 AF647

-CD21 BB700

DAPI (used at 1: 2000 final dilution)

Prolonggold sealant

Cover glass

Method

1. Selecting a glass slide:

the slide with the frozen section was removed from the-80 ℃ freezer.

The slides were warmed to room temperature and dried

2. Drawing slides with a DAKO PAP pen

3. Fixation with ice-cold acetone for 5min at room temperature. The acetone should be evaporated and the slide dried.

4. Simple washes with 1 × TBS

5. Sealing with 10% human AB serum sealant in a constant humidity chamber at room temperature for 10min

6. Ab mixtures were prepared in dilution buffer (containing 10% human serum).

7. The sealant is brushed off the groove.

8. Sections were incubated simultaneously with the Ab mixture for 1 hour at room temperature in a dark incubator

9. Brushing off Ab mixture

10. Briefly washed 1 time with TBS, followed by 3 times 5min each with TBS on a shaker

11. DAPI (final concentration 1: 2000) was added and incubated for 5min at room temperature in a dark incubator.

12. Wash 3 times, 2min each time, wash with TBS on shaker for ProlongGold mounting slides.

13. Imaging slides using Vectra Polaris

Results

Frozen tissue sections of melanoma-infiltrated lymph nodes were fixed with acetone, blocked with 0.25% casein + 10% human serum, and labeled with six antibodies (including anti-CD 31 BV480, anti-CD 141 BB515, anti-CD 3 AF532, anti-CD 34 PE-CF594, anti-Ki 67-AF647, and anti-CD 21 BB700) conjugated simultaneously with different fluorophores for 1 hour at room temperature. After washing, the tissue sections were then labeled with DAPI for 5min at room temperature and mounted. Subsequently, the tissue sections were scanned across the entire slide using a multispectral scanner from Vectra Polaris and a region of interest (ROI) was selected for multispectral imaging. The images obtained were unmixed in the InForm software (PerkinElmer) (fig. 1 to 6).

The ability of the methods disclosed herein to simultaneously detect the presence and abundance of multiple target antigens, biomarkers, and cell types is clearly illustrated in fig. 1-13, which show the distribution of different immune and stromal cell populations within melanoma infiltrated lymph node tissue sections simultaneously labeled with DAPI and six Ab-FPs as described herein.

Example 2

The following protocol is essentially the same as that set forth in example 1 for the specific detection of seven different target antigens in frozen sections of tumor tissue using the Ab-FP protocol described herein.

Following the inventive method described herein, multiple immunofluorescence images of eight colors were generated (7 Ab + DAPI).

As in example 1, the simultaneous addition of all Ab-FPs (seven) in this protocol, again illustrates the significant advantage disclosed herein, namely the greatly reduced total time required to specifically detect and identify multiple target antigens in a single sample.

This embodiment of the method of the invention also uses frozen tissue and therefore does not require deparaffinization and antigen retrieval.

Material

Frozen tissue slide

Ice-cold acetone

DAKO PAP pen

1X TBS

Constant humidity box

A sealing agent: 10% human serum prepared in 1 × TBS

Dilution buffer: 10% human serum prepared in 1 × TBS

Fluorophore conjugated antibodies

-CD31 BV480

-CD141 BB515

-CD3 AF532

-CD34 PE-CF594

-Ki67 AF647

-CD21 BB700

-CD11c AF700

DAPI (used at 1: 2000 final dilution)

Prolonggold sealant

Cover glass

Method

1. Selecting a glass slide:

the slide with the frozen section was removed from the-80 ℃ freezer.

The slides were warmed to room temperature and dried

2. Drawing slides with a DAKO PAP pen

4. Simple washes with 1 × TBS

5. Blocking with 10% human AB serum blocking agent in a constant humidity chamber at room temperature for 10min

6. Ab mixtures were prepared in dilution buffer (containing 10% human serum).

7. The sealant is brushed off the groove.

9. Brushing off Ab mixture

13. Imaging slides using Vectra Polaris

Results

Frozen tissue sections of melanoma-infiltrated lymph nodes were fixed with acetone, blocked with 0.25% casein + 10% human serum, and labeled with seven antibodies (including anti-CD 31 BV480, anti-CD 141 BB515, anti-CD 3 AF532, anti-CD 34 PE-CF594, anti-Ki 67-AF647, anti-CD 11c AF700, and anti-CD 21 BB700) conjugated simultaneously with different fluorophores for 1 hour at room temperature. After washing, the tissue sections were then labeled with DAPI for 5min at room temperature and mounted. Subsequently, the tissue sections were scanned across the entire slide using a multispectral scanner from Vectra Polaris and a region of interest (ROI) was selected for multispectral imaging. The images obtained were unmixed in InForm software (Perkinelmer) (FIGS. 14-23).

The ability of the methods disclosed herein to simultaneously detect the presence and abundance of multiple target antigens, biomarkers and cell types in frozen tissue sections is clearly illustrated in fig. 1-25, which show the distribution of different immune and stromal cell populations within melanoma infiltrating lymph node tissue sections labeled simultaneously with DAPI and six or seven Ab-FPs as described herein.

Example 3

The following protocol is essentially the same as set forth in example 1 for the specific detection of CD163+ cells in frozen sections of tumor tissue using the CD163 APC/Fire 750Ab-FP conjugates described herein.

Following the inventive methods described herein, a demixed immunofluorescence image is generated to further illustrate that the methods disclosed herein are capable of detecting Ab-FP conjugates that include FPs emitted at the far-infrared end of the spectrum.

As in example 1, the use of Ab-FP conjugates to directly label tissue further illustrates the significant advantage of the present invention, namely the identification of target antigens in tissue samples by the specific detection of antigen expression using a single Ab-FP conjugate for direct detection (as opposed to indirect or secondary detection).

Material

Frozen tissue slide

Ice cold acetone

DAKO PAP pen

1X TBS

Constant humidity box

A sealing agent: 10% human serum prepared in 1 × TBS

Dilution buffer: 10% human serum prepared in 1 × TBS

Fluorophore conjugated antibodies

-CD163 APC/Fire 750

Prolonggold sealant

Cover glass

Method

1. Selecting a glass slide:

the slide with the frozen section was removed from the-80 ℃ freezer.

The slides were warmed to room temperature and dried

2. Drawing slides with a DAKO PAP pen

4. Simple washes with 1 × TBS

6. Ab was prepared in dilution buffer (containing 10% human serum).

7. The sealant is brushed off the groove.

8. Sections were incubated simultaneously with Ab for 1 hour at room temperature in a dark incubator

9. Brushing off Ab

11. Glass slide mounting with Prolonggold

12. Imaging slides using Vectra Polaris

Results

Frozen tissue sections of melanoma-infiltrated lymph nodes were fixed with acetone, blocked with 0.25% casein + 10% human serum, and labeled with a single Ab-FP conjugate; anti-CD 163 APC/Fire 750 at room temperature for 1 hours. After washing, the tissue sections were mounted. Subsequently, the tissue sections were multi-spectral imaged using a multi-spectral scanner Vectra Polaris. The images obtained were unmixed in the InForm software (PerkinElmer) (fig. 24 and 25).

The methods disclosed herein are clearly illustrated in fig. 24 and 25 by the ability to detect the presence and abundance of target antigens, biomarkers and cell types by direct labeling using Ab-FP conjugates that include fluorophores that emit at the far infrared end of the spectrum as described herein. These figures show the distribution of CD163+ cell populations in melanoma infiltrating lymph node tissue sections.

Example 4

The following protocol was used to specifically detect two different target antigens in Formalin Fixed Paraffin Embedded (FFPE) tonsil tissue sections using Ab-FP as described herein. The following Ab-FP was used: CD45RO-AF488, CD19-AF647 and CD8-AF 647.

Following the inventive method described herein, a multiplex immunofluorescence image (2 Ab + DAPI) of three colors was generated.

Thus, following the methods described herein allows for rapid and specific multiplexed detection of multiple target antigens in FFPE tissue sections without the need for a secondary antibody incubation step.

The methods described herein allow for more rapid detection of marker co-localization in the same subcellular compartment than known multiplex immunofluorescence detection methods. The method also generates higher resolution images due to the reduced spread of fluorescence emission from the fluorophore in Ab-FP due to the direct conjugation of the fluorophore to the primary antibody.

In the method, two primary antibodies are added simultaneously in a one-step staining protocol. In contrast, following the Opal scheme, only a single one of the resists is added in a number of different steps to obtain a multi-color image. Thus, the methods described herein provide at least one significant advantage over known methods in that the total time required for specific immunofluorescence detection and identification of multiple target antigens in a single tissue section, particularly in FFPE tissue sections, is greatly reduced.

Materials and methods

Reagent

The following fluorophore-conjugated antibodies were purchased from Biolegend and BD Biosciences:

CD19 AF647

CD8 AF647

CD45RO AF488

CD45RO AF594

CD45RO AF700

CD45RO BV510

CD45RO BV650

CD45RO PE/Dazzle 594

CD45RO APC/Fire 750

standard materials used in the following examples:

positively charged glass slide (for tissue slice)

Cover glass

Baths and solvents for FFPE tissue dewaxing and rehydration

Restorer 2100 (for antigen restoration)

Xylene

Ethanol

H ₂ O

10％NBF

DAKO PAP pen

1X antigen retrieval buffer

1X TBS

Constant humidity box

A sealing agent: 0.25% Casein + 10% human serum prepared in 1 XTSS

Antibody dilution buffer: 10% human serum prepared in 1 × TBS

DAPI (used at 1: 2000 final dilution)

Prolonggold sealant

Tissue sample

Formalin fixed paraffin embedded tonsil tissues were provided by our clinical co-worker from the hospital of auckland city.

Tissue preparation and staining protocol

(1) Baking the slides in an oven set at 60 ℃ for at least 1 hour until overnight

(2) Dewaxing and rehydration: the slides were loaded into the staining rack and the following treatments were performed:

xylene: 3 times for 10min each

99% ethanol: 2 times, each for 5min

90% EtOH: 1 time for 10min

70% EtOH: 30 seconds

dH 2O: 30 seconds

10% NBF: 1 time for 10min

dH 2O: 30 seconds

(3) Slide glass is loaded into slide chamber containing 1X antigen retrieval buffer

(4) The slide chamber is placed into the repairer 2100 filled with dH 2O. Press down to start

(5) After cooling the slides, wash 5 times with dH2O and twice in 1 × TBS, each for 5 min.

(6) The liquid surrounding the slices was wiped with a paper towel. Circle the section with PAP pen to limit the area

(7) Tissue sections were incubated with 10% HS blocking agent for 10min at room temperature in a humidified chamber

(8) Brushing off sealant

(9) Fluorophore conjugated antibodies prepared in antibody dilution buffer were added simultaneously to the tissue sections and incubated for 1 hour at room temperature in a dark incubator (protected from light).

(10) Ab was brushed off on the trough. Briefly washed 1 time with TBS, followed by 3 times 5min each with TBS on a shaker

(11) DAPI (1: 2000) was added to the tissue sections and incubated for 5min at room temperature in a dark incubator (protected from light).

(12) Wash 3 times, 2min each time, wash with TBS on a shaker and seal slides with coverslips using prolong gold.

(13) Imaging was continued using Vectra Polaris.

Scanning protocol (from Vectra Polaris instruction 1.0.7)

(1) Opening Vectra Polaris Instrument and computer

(2) Launching Vectra Polaris software

(3) Loading slides into the stage

(5) In the "edit plan" page (Vectra Polaris software), an imaging plan is created. The fluorescence mode and spatial resolution (typically x20 magnification, x10 or x40 are also available) were chosen for multi-spectral imaging (MSI) of the entire slide scan (WSS) and region of interest (ROI). The exposure times for the WSS and MSI and filters for focusing and imaging are also set.

Results

Formalin Fixed Paraffin Embedded (FFPE) tissue sections from tonsils were deparaffinized in xylene, rehydrated, and heat treated in a reconditioner with antigen reconditioning buffer. The tissue sections were then blocked with 0.25% casein + 10% human serum and labeled with Ab-FP conjugate and DAPI. Subsequently, the tissue sections were multi-spectral imaged using a multi-spectral scanner Vectra Polaris. The images obtained were unmixed in the InForm software (Perkinelmer).

The ability of the methods disclosed herein to simultaneously detect the presence and abundance of multiple target antigens, biomarkers and cell types in FFPE tissue sections is clearly illustrated in fig. 26-36, which show the distribution of antigen and cell populations in tonsil tissue sections labeled simultaneously with DAPI and one or two Ab-FPs as described herein, in fig. 26-36.

Industrial applicability

The antibody-fluorophore conjugates of the invention and methods of using the same have industrial utility in molecular biology, providing a means of diagnosing and managing diseases, including cancer.

Reference to the literature

Gorriset al.MAJ,.(2018).Eight-Color Multiplex Immunohistochemistry for Simultaneous Detection of Multiple Immune Checkpoint Molecules within the Tumor Microenvironment.J Immunol.,200(1),347-354.

Hofmanet al.Paul,.(2019).Multiplexed Immunohistochemistry for Molecular and Immune Profiling in Lung Cancer—Just About Ready for Prime-TimeCancers(Basel),11(3):283.

Majtahedet al.Amirkaveh,.(2011).A two-antibody mismatch repair protein immunohistochemistry screening approach for colorectal carcinomas,skin sebaceous tumors,and gynecologic tract carcinomas.Modern Pathology,24,1004-1014.

Soodet al.Anup,.(2016).Multiplexed immunofluorescence delineates proteomic cancer cell states associated with metabolism.JCI Insight,1(6).

Claims

1. A composition comprising at least three, four, five, six, or at least seven antibody-fluorophore conjugates (Ab-FP), wherein each FP has a different fluorescence excitation and emission spectrum (Ex).

2. The composition of claim 1, comprising seven Ab-FPs.

3. The composition of claim 1 or claim 2, wherein each Ab-FP is a unique Ab-FP.

4. The composition of any one of claims 1 to 3, wherein the composition comprises at least eight, nine, ten, eleven, or twelve Ab-FP.

5. The composition according to any one of claims 1 to 4, wherein the maximum excitation and emission wavelength (Ex/Em) of each FP is selected from the group consisting of 348/395, 404/448, 405/421, 405/510, 405/570, 405/603, 405/646, 405/711, 407/421, 415/500, 436/478nm, 490/515nm, 494/520nm, 495/519nm, 485/693nm, 496/578, 532/554nm, 566/610nm, 590/620nm, 650/660nm, 650/668nm, 652/704, 696/719nm, 753/785nm, 754/787nm, 755/775nm and 759/775 nm.

6. The composition according to any one of claims 1-5, wherein the maximum fluorescence emission wavelength (Em) of at least one, two, or three of the FPs is from about 710nm to about 850 nm.

7. The composition of claim 6, wherein the Em of at least one, two, or three of the FPs is from about 753nm to about 759nm, preferably 753nm, 754nm, 755nm, or 759 nm.

8. The composition according to any one of claims 1 to 7, wherein the FP is selected from the group consisting of Brilliant with Ex/Em of 348/395 ^TM Ultraviolet 395(BUV395), Brilliant with Ex/Em of 436/478nm ^TM Violet 480(BV480), Brilliant Violet 421 with Ex/Em 405/421 ^TM Brilliant with Ex/Em of 407/421 ^TM Brilliant with Violet 421(BV421), Ex/Em 405/510 ^TM Violet 510(BV510), Brilliant Violet 570 with Ex/Em 405/570 ^TM Brilliant Violet 605 with Ex/Em of 405/603 ^TM Brilliant Violet 650 with Ex/Em of 405/646 ^TM Brilliant Violet 711 with Ex/Em of 405/711 ^TM BD Horizon with Ex/Em of 404/448 ^TM BD Horizon with V450 and Ex/Em of 415/500 ^TM Brilliant with V500 and Ex/Em of 490/515nm ^TM Blue 515(BB515), Fluorescein Isothiocyanate (FITC) with Ex/Em of 494/520nm, Alexa Fluor 488(AF488) with Ex/Em of 495/519nm, Alexa Fluor 532(AF532) with Ex/Em of 532/554nm, R-Phycoerythrin (PE) with Ex/Em of 496/578, Alexa Fluor (AF594) with Ex/Em of 590/620nm, PE-Dazzle 594(PE594) or PE-CF594(CF594) with Ex/Em of 566/610nm, Alexa Fluor 647(AF647) with Ex/Em of 650/668nm, Allophycocyanin (APC) with Ex/Em of 650/660nm, BD Horizon of 485/693nm ^TM 700(BB700), Alexa Fluor 700(AF700) with Ex/Em of 696/719nm, APC/Alexa Fluor 750 with Ex/Em of 753/785nm, APC/Fire 750 with Ex/Em of 754/787nm, APC-R700 with Ex/Em of 652/704, APC-Cy7 with Ex/Em of 755/775nm, and AF750 with Ex/Em of 759/775 nm.

9. The composition of any one of claims 1 to 8, wherein the fluorophore is selected from the group consisting of BV480, BB515, AF532, PE-CF594, AF647, AF700 and BB 700.

10. The composition of any one of claims 1 to 9, wherein the Ab in the Ab-FP is selected from the group consisting of anti-CD 31, CD141, CD144, CD3, CD34, CD163, CD11c, CD14, CD16, CD68, Foxp3, CD4, CD8, CD19, CD20, CD25, CD38, PD-1, PDL1, PDL2, CD68, Ki-67, Sox10, S100, PRAME, MART1, and anti-CD 21 antibodies.

11. The composition of any one of claims 1 to 10, wherein the Ab in the Ab-FP is selected from the group consisting of anti-CD 31, CD141, CD3, CD34, Ki-67, CD11c, and anti-CD 21 antibodies.

12. The composition according to any one of claims 1 to 11, comprising at least three, preferably four, five, six or preferably all seven of the following antibodies: anti-CD 31, CD141, CD3, CD34, Ki-67, CD11c, and anti-CD 21 antibodies.

13. The composition of any one of claims 1 to 12, comprising at least three, preferably four, five, six, or preferably all seven of the following Ab-FPs: CD31-BV 480; CD141-BB 515; CD3-AF 532; CD34-PE-CF 594; ki67-AF647, CD11c-AF700 and CD21-BB 700.

14. The composition of any one of claims 1-9, wherein the Ab in the Ab-FP comprises one or more Ab selected from the group consisting of anti-Estrogen Receptor (ER), Progesterone Receptor (PR), her2, and anti-cytokeratin antibody.

15. The composition of any one of claims 1-9, wherein the abs in the Ab-FP comprise one or more anti-mismatch repair protein antibodies or anti-mutant mismatch repair protein antibodies.

16. The composition of any one of claims 1 to 9, wherein the Ab in the Ab-FP comprises one or more antibodies selected from the group consisting of anti-b-raf (V600E mutation), MLH1, MSH2, MSH6, and anti-PMS 2 antibodies.

17. The composition of any one of claims 1 to 9, wherein each Ab specifically binds a target antigen.

18. The composition of claim 17, wherein each target antigen is a biomarker for a protein or cell type, preferably a different protein or a different cell type.

19. The composition of claim 17 or claim 18, wherein each target antigen is a biomarker for a cell surface receptor or intracellular antigen.

20. The composition of any one of claims 17 to 19, wherein each target antigen is a T cell, B cell, macrophage, monocyte, or dendritic cell antigen.

21. The composition of any one of claims 17 to 20, wherein each target antigen is a T cell antigen selected from the group consisting of CD3, CD4, CD8, FoxP3, CD25, CD137, CD38, CD69, PD-1, CTLA-4, and Ki67 antigens.

22. The composition of any one of claims 17 to 20, wherein each target antigen is a B cell antigen selected from the group consisting of CD19, CD20, CD21, BCL-6, BLIMP1, Ki67, and CD138 antigens.

23. The composition of any one of claims 17 to 20, wherein each target antigen is a macrophage or monocyte antigen selected from the group consisting of CD14, CD16, CD68, and CD163 antigens.

24. The composition of any one of claims 17 to 20, wherein each target antigen is a dendritic cell antigen, CD1c or CLEC9a antigen.

25. A method of direct immunofluorescence analysis of a biological sample, comprising:

a. labeling at least one target antigen in a planar biological sample with at least one unique Ab-FP conjugate, and

b. generating a multispectral fluorescence image of the labeled planar sample using a multispectral scanner, wherein the image comprises at least two colors, wherein at least one color is associated with the specific binding of the at least one unique Ab-FP conjugate and the at least one target antigen, and

c. determining from the image the presence or absence of at least one biomarker comprising the at least one target antigen.

26. The method of claim 25, wherein labeling in a) comprises simultaneously labeling at least two, preferably at least three, four, five, six, preferably at least seven different target antigens with at least two, preferably at least three, four, five, six, preferably at least seven unique Ab-FP conjugates.

27. The method of claim 25 or claim 26, wherein labeling in a) comprises simultaneous labeling with seven unique Ab-FP conjugates.

28. The method according to any one of claims 25 to 27, wherein one color in b) is correlated with the fluorescence emission spectrum of a nuclear stain, preferably DAPI or Hoechst 33342.

29. The method according to any one of claims 25 to 28, wherein the multispectral image in b) comprises at least three, preferably at least four, five, six, preferably seven different colors, wherein each different color is associated with the specific binding of Ab-FP and target antigen.

30. The method of any one of claims 25 to 29, wherein the generating the multispectral image in b) comprises separately detecting at least two, preferably three, four, five, six, preferably seven different fluorescence spectra using the multispectral scanner, wherein each detected spectrum corresponds to an Ab-FP that specifically binds to a target antigen.

31. The method according to any one of claims 25 to 30, wherein the planar biological sample is a tissue section, preferably a frozen tissue section.

32. The method of claim 31, wherein the tissue section is from a mammal, preferably a human.

33. The method of claim 31 or 32, wherein the tissue section is from liver, skin, lung, breast, colon, or lymph node.

34. The method of any one of claims 25 to 33, wherein the target antigen is as defined in any one of claims 18 to 24.

35. The method of any one of claims 25 to 33, wherein Ab is as defined in any one of claims 10 to 12 and 14 to 17.

36. The method according to any one of claims 25 to 35, wherein the labeling in a) comprises labeling with at least two, preferably three, four, five, six or preferably seven of the following antibodies: anti-CD 31, CD141, CD3, CD34, Ki67, CD11c and anti-CD 21 antibodies.

37. The method of any one of claims 25 to 36, wherein labeling in a) comprises labeling with at least two, preferably three, four, five, or preferably all six of the following Ab-FP: CD31-BV 480; CD141-BB 515; CD3-AF 532; CD34-PE-CF 594; ki67-AF 647; CD11c-AF700 and CD21-BB 700.

38. The method of claim 36 or claim 37, wherein labeling in a) further comprises replacing one Ab-FP with a different Ab-FP, wherein the replacement Ab-FP comprises the same Ab and an FP with an Ex/Em of about 753nm to about 759nm, preferably 753nm, 754nm, 755nm, or 759 nm.

39. The method according to any one of claims 25 to 37, wherein labeling in a) further comprises labeling an additional target antigen with an additional Ab-FP comprising an FP having an Ex/Em of about 753nm to about 759nm, preferably 753nm, 754nm, 755nm, or 759 nm.

40. The method of claim 39, wherein the additional Ab-FP comprises an anti-CD 31, CD141, CD3, CD34, Ki-67, CD11c, or anti-CD 21 Ab.

41. The method of claim 39, wherein the additional Ab does not comprise an anti-CD 31, CD141, CD3, CD34, Ki-67, CD11c, or anti-CD 21 Ab.

42. The method according to any one of claims 25 to 40, wherein the determination in c) is the detection of the presence of at least two, preferably at least three, four, five, six, preferably at least seven different biomarkers.

43. The method of any one of claims 25 to 42, wherein the determination in c) comprises determining the abundance, preferably the relative abundance, of at least two, preferably at least three, four, five, six, preferably at least seven different biomarkers.

44. The method of any one of claims 25 to 43, wherein the determination in c) comprises detecting one or more biomarkers on or in cells of the immune system, wherein the cells of the immune system are selected from the group consisting of T cells, B cells, macrophages, monocytes and dendritic cells.

45. The method of any one of claims 25 to 33, 42 and 43, wherein the determination in c) comprises detecting one or more biomarkers on or in at least one tumor cell.

46. The method of any one of claims 25 to 43, wherein the determination in c) comprises detecting clinically relevant levels of one or more biomarkers.

47. The method according to any one of claims 25-46, wherein the multispectral image in b) is generated by computer image analysis of fluorescence intensity data from each pixel in the image captured by the multispectral scanner.

48. The method according to any one of claims 26 to 47, wherein the image generated in b) comprises at least two distinct colors, preferably at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, preferably twelve distinct colors, wherein each color is associated with a different FP fluorescence emission spectrum.

49. The method of any one of claims 26 to 48, wherein the image generated in b) comprises at least four, preferably at least five, six, seven or eight unique colors, wherein each color is associated with a different FP fluorescence emission spectrum.

50. The method of claim 49, wherein the image generated in b) comprises at least six or seven unique colors.

51. The method of claim 50, wherein at least one, two, or three unique colors specifically correspond to FP emission spectra of about 710 and about 850nm, preferably corresponding to FP maximum emission wavelengths of about 753nm to about 759nm, preferably 753nm, 754nm, 755nm, or 759 nm.